Controlled release auris sensory cell modulator compositions and methods for the treatment of otic disorders

ABSTRACT

Disclosed herein are compositions and methods for the treatment of otic diseases or conditions with auris sensory cell modulating agent compositions and formulations administered locally to an individual afflicted with an otic disease or condition, through direct application of these compositions and formulations onto or via perfusion into the targeted auris structure(s).

CROSS-REFERENCE

This patent application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/082,450 filed Jul. 21, 2008; U.S. ProvisionalApplication Ser. No. 61/083,830 filed Jul. 25, 2008; U.S. ProvisionalApplication Ser. No. 61/086,094, filed Aug. 4, 2008; U.S. ProvisionalApplication Ser. No. 61/094,384 filed Sep. 4, 2008; U.S. ProvisionalApplication Ser. No. 61/101,112 filed Sep. 29, 2008; U.S. ProvisionalApplication Ser. No. 61/140,033 filed Dec. 22, 2008; U.S. ProvisionalApplication Ser. No. 61/160,233, filed Mar. 13, 2009; U.S. ProvisionalApplication Ser. No. 61/164,812, filed Mar. 30, 2009; U.S. ProvisionalApplication Ser. No. 61/174,421 filed Apr. 30, 2009; and UK PatentApplication No. 0907070.7, filed Apr. 24, 2009; all of which areincorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Vertebrates have a pair of ears, placed symmetrically on opposite sidesof the head. The ear serves as both the sense organ that detects soundand the organ that maintains balance and body position. The ear isgenerally divided into three portions: the outer ear, auris media (ormiddle ear) and the auris interna (or inner ear).

SUMMARY OF THE INVENTION

Described herein are compositions, formulations, manufacturing methods,therapeutic methods, uses, kits, and delivery devices for the controlledrelease or delivery of at least one auris sensory cell modulator to atleast one structure or region of the ear. In some embodiments, an aurissensory cell modulator is an auris sensory cell damage agent. In someembodiments, an auris sensory cell modulator is an auris sensory celldeath agent. In some embodiments, an auris sensory cell modulator is anauris sensory cell protection agent (e.g., promotes survival of aurissensory cells). In some embodiments, an auris sensory cell modulator isan auris sensory cell growth/regeneration agent. Disclosed herein arecontrolled release compositions for treating or ameliorating hearingloss or reduction resulting from destroyed, stunted, malfunctioning,damaged, fragile or missing hairs in the inner ear. In one embodiment,the controlled release composition comprises a therapeutically-effectiveamount of at least one modulator of neuron and/or hair cells of theauris (also known as “auris sensory cell modulating agent”), acontrolled release auris-acceptable excipient, and an auris-acceptablevehicle.

In some embodiments, the target portion of the ear is the middle ear orauris media. In other embodiments, the target portion of the ear is theinner ear, or auris interna. In other embodiments, the target portion ofthe ear is the middle ear, or auris media. In still other embodiments,the target portion of the ear is both the auris media and the aurisinterna. In some embodiments, the controlled release formulationsfurther comprise a rapid or immediate release component for deliveringan auris sensory cell modulating agent to the auris media and/or theauris interna. All formulations comprise excipients that are auris-mediaand/or auris-interna acceptable.

Described herein are methods for inducing selective auris sensory celldamage and/or death within the auris media or auris interna comprisingadministration of the auris sensory cell modulating agent controlledrelease formulations described herein. Also described herein are methodsfor inducing auris sensory cell growth and/or reversal of damage toauris sensory cells (e.g. stunted or malfunctioning auris sensory cells)and/or protection against further damage to auris sensory cells (e.g.stunted or malfunctioning auris sensory cells) comprising administrationof the auis sensory cell modulating agent compositions described herein.

Also disclosed herein are methods for the treatment of otic disorderscomprising administration of auris sensory cell modulating agentcontrolled release formulations. In some embodiments, the auris sensorycell modulating agent is a toxicant (e.g., gentamicin) and induces celldeath. In some embodiments, the auris sensory cell modulating agent isan otoprotectant, e.g., amifostine, an antioxidant, or the like. In someembodiments, the auris sensory cell modulating agent induces growth ofnew auris sensory cells, e.g., an adenovirus expressing Atoh1. In someembodiments, the auris sensory cell modulating is an agent that reducesor inhibits auris sensory cell death, e.g., a glutamate receptormodulator.

In some embodiments, a glutamate receptor modulator is a glutamatereceptor antagonist. In some embodiments, a glutamate receptorantagonist is an NMDA receptor antagonist. In some embodiments, theagent that modulates the NMDA receptor is an NMDA receptor antagonist.In some embodiments, an auris sensory cell modulating agent is a trophicagent (e.g., an agent that promotes growth of healthy cells and/ortissue). In some embodiments, a trophic agent is a growth factor. Insome embodiments, a growth factor is brain-derived neurotrophic factor(BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derivedneurotrophic factor (GDNF), neurotrophin-3, neurotrophin-4, fibroblastgrowth factor (FGF), insulin-like growth factor (IGF) or the like and/orcombinations thereof. In some embodiments, the auris sensory cellmodulating agent is an agent that has a protective effect, e.g., immunesystem cells that reduce inflammation and/or infections. In someembodiments, the immune system cells are macrophages, microglia, and/ormicroglia-like cells. In some embodiments, the auris sensory cellmodulating agent is a Na+ channel blocker and reduces or inhibits aurissensory cell damage and/or death. In some embodiments, the auris sensorycell modulating agent is an otoprotectant, e.g., a corticosteroid thatreduces, delays or reverses damage to auris sensory cells. In someembodiments, the auris sensory cell modulating agent is an ototoxicagent or a toxicant that causes auris sensory cell death. In someembodiments, the auris sensory cell modulating agent is a modulator ofpRB. In some embodiments, the auris sensory cell modulating agent is amodulator of a TH receptor. In some embodiments, the auris sensory cellmodulating agent is an adenovirus expressing a BRN3. In someembodiments, the auris sensory cell modulating agent is an agonist of aBRN3. In some embodiments, the auris sensory cell modulating agent is anantagonist of a BRN3. In some embodiments, the auris sensory cellmodulating agent is a stem cell and/or differentiated auris sensory cellthat promotes growth and/or regeneration of auris sensory cells.

In some embodiments, the composition further comprises an auris sensorycell modulating agent as an immediate release agent wherein theimmediate release auris sensory cell modulating agent is the same agentas the controlled-release agent, a different auris sensory cellmodulating agent, an additional therapeutic agent, or a combinationthereof.

In some embodiments, the composition further comprises an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis an anesthetic, a local acting anesthetic, an analgesic, anantibiotic, an antiemetic, an antifungal, an anti-microbial agent, anantiseptic, an antiviral, a chemotherapeutic agent, a diuretic, akeratolytic agent, an otoprotectant (e.g., a steroid), animmunosuppressant comprising but not limited to such agents ascalcineurin inhibitors (cyclosporine, tacrolimus, pimecrolimus),macrolides such as rapamycin, corticosteroids, or combinations thereof.In some embodiments, the additional therapeutic agent is an immediaterelease agent. In some embodiments, the additional therapeutic agent isa controlled release agent.

Disclosed herein are controlled release formulations for delivering anauris sensory cell modulating agent to the ear. In some embodiments, thecomposition is administered so that the composition is in contact withthe crista fenestrae cochleae, the round window or the tympanic cavity.

The auris formulations and therapeutic methods described herein havenumerous advantages that overcome the previously-unrecognizedlimitations of formulations and therapeutic methods described in priorart.

Sterility

The environment of the inner ear is an isolated environment. Theendolymph and the perilymph are static fluids and are not in contiguouscontact with the circulatory system. The blood—labyrinth—barrier (BLB),which includes a blood-endolymph barrier and a blood-perilymph barrier,consists of tight junctions between specialized epithelial cells in thelabyrinth spaces (i.e., the vestibular and cochlear spaces). Thepresence of the BLB limits delivery of active agents (e.g., aurissensory cell modulating agents) to the isolated microenvironment of theinner ear. Auris hair cells are bathed in endolymphatic or perilymphaticfluids and cochlear recycling of potassium ions is important for haircell function. When the inner ear is infected, there is an influx ofleukocytes and/or immunoglobins (e.g. in response to a microbialinfection) into the endolymph and/or the perilymph and the delicateionic composition of inner ear fluids is upset by the influx ofleukocytes and/or immunoglobins. In certain instances, a change in theionic composition of inner ear fluids results in hearing loss, loss ofbalance and/or ossification of auditory structures. In certaininstances, even trace amounts of pyrogens and/or microbes can triggerinfections and related physiological changes in the isolatedmicroenvironment of the inner ear.

Due to the susceptibility of the inner ear to infections, aurisformulations require a level of sterility (e.g., low bioburden) that hasnot been recognized hitherto in prior art. Provided herein are aurisformulations that are manufactured with low bioburden or sterilized withstringent sterility requirements and are suitable for administration tothe middle and/or inner ear. In some embodiments, the auris compatiblecompositions described herein are substantially free of pyrogens and/ormicrobes.

Compatibility with Inner Ear Environment

Described herein are otic formulations with an ionic balance that iscompatible with the perilymph and/or the endolymph and does not causeany change in cochlear potential. In specific embodiments,osmolarity/osmolality of the present formulations is adjusted, forexample, by the use of appropriate salt concentrations (e.g.,concentration of sodium salts) or the use of tonicity agents whichrenders the formulations endolymph-compatible and/orperilymph-compatible (i.e. isotonic with the endolymph and/orperilymph). In some instances, the endolymph-compatible and/orperilymph-compatible formulations described herein cause minimaldisturbance to the environment of the inner ear and cause minimumdiscomfort (e.g., vertigo) to a mammal (e.g., a human) uponadministration. Further, the formulations comprise polymers that arebiodegradable and/or dispersable, and/or otherwise non-toxic to theinner ear environment. In some embodiments, the formulations describedherein are free of preservatives and cause minimal disturbance (e.g.,change in pH or osmolarity, irritation) in auditory structures. In someembodiments, the formulations described herein comprise antioxidantsthat are non-irritating and/or non-toxic to otic structures.

Dosing Frequency

The current standard of care for auris formulations requires multipleadministrations of drops or injections (e.g. intratympanic injections)over several days (e.g., up to two weeks), including schedules ofreceiving multiple injections per day. In some embodiments, aurisformulations described herein are controlled release formulations, andare administered at reduced dosing frequency compared to the currentstandard of care. In certain instances, when an auris formulation isadministered via intratympanic injection, a reduced frequency ofadministration alleviates discomfort caused by multiple intratympanicinjections in individuals undergoing treatment for a middle and/or innerear disease, disorder or condition. In certain instances, a reducedfrequency of administration of intratympanic injections reduces the riskof permanent damage (e.g., perforation) to the ear drum. Theformulations described herein provide a constant, sustained, extended,delayed or pulsatile rate of release of an active agent into the innerear environment and thus avoid any variability in drug exposure intreatment of otic disorders.

Therapeutic Index

Auris formulations described herein are administered into the ear canal,or in the vestibule of the ear. Access to, for example, the vestibularand cochlear apparatus will occur through the auris media including theround window membrane, the oval window/stapes footplate, the annularligament and through the otic capsule/temporal bone. Otic administrationof the formulations described herein avoids toxicity associated withsystemic administration (e.g., hepatotoxicity, cardiotoxicity,gastrointestinal side effects, renal toxicity) of the active agents. Insome instances, localized administration in the ear allows an activeagent to reach a target organ (e.g., inner ear) in the absence ofsystemic accumulation of the active agent. In some instances, localadministration to the ear provides a higher therapeutic index for anactive agent that would otherwise have dose-limiting systemic toxicity.

Prevention of Drainage into Eustachian Tube

In some instances, a disadvantage of liquid formulations is theirpropensity to drip into the eustachian tube and cause rapid clearance ofthe formulation from the inner ear. Provided herein, in certainembodiments, are auris formulations comprising polymers that gel at bodytemperature and remain in contact with the target auditory surfaces(e.g., the round window) for extended periods of time. In someembodiments, the formulations further comprise mucoadhesives that allowthe formulations to adhere to otic mucosal surfaces. In some instances,the auris formulations described herein avoid attenuation of therapeuticbenefit due to drainage or leakage of active agents via the eustachiantube.

DESCRIPTION OF CERTAIN EMBODIMENTS

Described herein are controlled release compositions and devices fortreating otic disorders comprising a therapeutically-effective amount ofan auris sensory cell modulating agent, a controlled releaseauris-acceptable excipient and an auris-acceptable vehicle. In oneaspect, the controlled release auris-acceptable excipient is chosen froman auris-acceptable polymer, an auris-acceptable viscosity enhancingagent, an auris-acceptable gel, an auris-acceptable paint, anauris-acceptable foam, an auris-acceptable microsphere or microparticle,an auris-acceptable hydrogel, an auris-acceptable in situ forming spongymaterial, an auris-acceptable actinic radiation curable gel, anauris-acceptable liposome, an auris-acceptable nanocapsule ornanosphere, an auris-acceptable thermoreversible gel or combinationsthereof. In further embodiments, the auris-acceptable viscosityenhancing agent is a cellulose, a cellulose ether, alginate,polyvinylpyrrolidone, a gum, a cellulosic polymer or combinationsthereof. In yet another embodiment, the auris-acceptable viscosityenhancing agent is present in an amount sufficient to provide aviscosity of between about 1000 to about 1,000,000 centipoise. In stillanother aspect, the auris-acceptable viscosity enhancing agent ispresent in an amount sufficient to provide a viscosity of between about50,000 to about 1,000,000 centipoise. In some embodiments, the aurissensory cell modulating agent formulations or compositions are optimalfor osmolality or osmolarity of the target auris structure to ensurehomeostasis is maintained.

In some embodiments, the compositions are formulated for pH, and apractical osmolality or osmolarity to ensure that homeostasis of thetarget auris structure is maintained. A perilymph-suitableosmolarity/osmolality is a practical/deliverable osmolarity/osmolalitythat maintains the homeostasis of the target auris structure duringadministration of the pharmaceutical formulations described herein.

For example, the osmolarity of the perilymph is between about 270-300mOsm/L, and the compositions described herein are optionally formulatedto provide a practical osmolarity of about 150 to about 1000 mOsm/L. Incertain embodiments, the formulations described herein provide apractical and/or deliverable osmolarity within about 150 to about 500mOsm/L at the target site of action (e.g., the inner ear and/or theperilymph and/or the endolymph). In certain embodiments, theformulations described herein provide a practical osmolarity withinabout 200 to about 400 mOsm/L at the target site of action (e.g., theinner ear and/or the perilymph and/or the endolymph). In certainembodiments, the formulations described herein provide a practicalosmolarity within about 250 to about 320 mOsm/L at the target site ofaction (e.g., the inner ear and/or the perilymph and/or the endolymph).In certain embodiments, the formulations described herein provide aperilymph-suitable osmolarity within about 150 to about 500 mOsm/L,about 200 to about 400 mOsm/L or about 250 to about 320 mOsm/L at thetarget site of action (e.g., the inner ear and/or the perilymph and/orthe endolymph). In certain embodiments, the formulations describedherein provide a perilymph-suitable osmolality within about 150 to about500 mOsm/kg, about 200 to about 400 mOsm/kg or about 250 to about 320mOsm/kg at the target site of action (e.g., the inner ear and/or theperilymph and/or the endolymph). Similarly, the pH of the perilymph isabout 7.2-7.4, and the pH of the present formulations is formulated(e.g., with the use of buffers) to provide a perilymph-suitable pH ofabout 5.5 to about 9.0, about 6.0 to about 8.0 or about 7.0 to about7.6. In certain embodiments, the pH of the formulations is within about6.0 to about 7.6. In certain instances, the pH of the endolymph is about7.2-7.9, and the pH of the present formulations is formulated (e.g.,with the use of buffers) to be within about 5.5 to about 9.0, withinabout 6.5 to about 8.0 or within about 7.0 to about 7.6.

In some aspects, the controlled-release auris-acceptable excipient isbiodegradable. In some aspects the controlled release auris-acceptableexcipient is bioeliminated (e.g., degraded and/or eliminated throughurine, feces or other routes of elimination). In another aspect, thecontrolled release composition further comprises an auris-acceptablemucoadhesive, an auris-acceptable penetration enhancer or anauris-acceptable bioadhesive.

In one aspect, the controlled release auris sensory cell modulatingagent composition is delivered using a drug delivery device, which is aneedle and syringe, a pump, a microinjection device or combinationsthereof. In some embodiments, the auris sensory cell modulating agent ofthe controlled release composition has limited or no systemic release,is toxic when administered systemically, has poor pK characteristics orcombinations thereof. In some aspects, the auris sensory cell modulatingagent is a small molecule agent. In other aspects, the auris sensorycell modulating agent is an antibody.

Also disclosed herein is a method for treating an otic disordercomprising administering at least once every 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 days, at least once a week, once every two weeks,once every three weeks, once every four weeks, once every five weeks, oronce every six weeks; or once a month, once every two months, once everythree months, once every four months, once every five months, once everysix months, once every seven months, once every eight months, once everynine months, once every ten months, once every eleven months, or onceevery twelve months with the compositions and formulations disclosedherein. In particular embodiments, the controlled release formulationsdescribed herein provide a sustained dose of auris sensory cellmodulating agent to the inner ear between subsequent doses of thecontrolled release formulation. That is, taking one example only, if newdoses of the auris sensory cell modulating agent controlled releaseformulation are adminstered via intratympanic injection to the roundwindow membrane every 10 days, then the controlled release formulationprovides an effective dose of auris sensory cell modulating agent to theinner ear (e.g., across the round window membrane) during that 10-dayperiod.

In one aspect, the composition is administered so that the compositionis in contact with the crista fenestrae cochleae, the round windowmembrane or the tympanic cavity. In one aspect the composition isadministered by intratympanic injection.

Provided herein, in some embodiments, are methods of selectivelyinducing auris sensory cell damage comprising administering to anindividual in need thereof an intratympanic composition or devicecomprising a therapeutically effective amount of an ototoxic agent, thecomposition or device comprising substantially low degradation productsof the ototoxic agent, the composition or device further comprising twoor more characteristics selected from:

-   -   (i) between about 0.1% to about 10% by weight of the ototoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) sterile water, q.s., buffered to provide a pH between        about 5.5 and about 8.0;    -   (iv) multiparticulate ototoxic agent;    -   (v) a gelation temperature between about 19° C. to about 42° C.;    -   (vi) less than about 50 colony forming units (cfu) of        microbiological agents per gram of formulation;    -   (vii) less than about 5 endotoxin units (EU) per kg of body        weight of a subject;    -   (viii) a mean dissolution time of about 30 hours for the        ototoxic agent; and    -   (ix) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments of the methods, the ototoxic agent is released fromthe composition or device for a period of at least 3 days. In someembodiments of the methods, the ototoxic agent is released from thecomposition or device for a period of at least 5 days. In someembodiments of the methods, the ototoxic agent is essentially in theform of micronized particles.

Also provided herein are methods of inducing auris sensory cell growthcomprising administering to an individual in need thereof anintratympanic composition or device comprising a therapeuticallyeffective amount of a trophic agent, the composition or devicecomprising substantially low degradation products of the trophic agent,the composition or device further comprising two or more characteristicsselected from:

-   -   (i) between about 0.1% to about 10% by weight of the trophic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) sterile water, q.s., buffered to provide a pH between        about 5.5 and about 8.0;    -   (iv) multiparticulate trophic agent;    -   (v) a gelation temperature between about 19° C. to about 42° C.;    -   (vi) less than about 50 colony forming units (cfu) of        microbiological agents per gram of formulation;    -   (vii) less than about 5 endotoxin units (EU) per kg of body        weight of a subject;    -   (viii) a mean dissolution time of about 30 hours for the trophic        agent; and    -   (ix) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments of the methods, the trophic agent is released fromthe composition or device for a period of at least 3 days. In someembodiments of the methods, the trophic agent is released from thecomposition or device for a period of at least 5 days. In someembodiments of the methods, the trophic agent is essentially in the formof micronized particles.

Also provided herein are methods of alleviating damage to auris sensorycells from an otic intervention comprising administering to anindividual in need thereof an intratympanic composition or devicecomprising a therapeutically effective amount of an otoprotectant, thecomposition or device comprising substantially low degradation productsof the otoprotectant, the composition or device further comprising twoor more characteristics selected from:

-   -   (i) between about 0.1% to about 10% by weight of the        otoprotectant, or pharmaceutically acceptable prodrug or salt        thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) sterile water, q.s., buffered to provide a pH between        about 5.5 and about 8.0;    -   (iv) multiparticulate otoprotectant agent;    -   (v) a gelation temperature between about 19° C. to about 42° C.;    -   (vi) less than about 50 colony forming units (cfu) of        microbiological agents per gram of formulation;    -   (vii) less than about 5 endotoxin units (EU) per kg of body        weight of a subject;    -   (viii) a mean dissolution time of about 30 hours for the        otoprotectant agent; and    -   (ix) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments of the methods, the otoprotectant is administeredbefore or during an otic intervention. In some embodiments of themethods, the otoprotectant is administered after an otic intervention.In some embodiments of the methods, the otoprotectant is essentially inthe form of micronized particles.

In some embodiments of the methods described above, the composition ordevice comprises:

-   -   (i) between about 0.1% to about 10% by weight of the auris        sensory cell modulating agent, or pharmaceutically acceptable        prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) multiparticulate auris sensory cell modulating agent;    -   (iv) a gelation temperature between about 19° C. to about 42°        C.; and    -   (v) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments of the methods described above, the composition ordevice comprises:

-   -   (i) between about 0.1% to about 10% by weight of the auris        sensory cell modulating agent, or pharmaceutically acceptable        prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) multiparticulate auris sensory cell modulating agent;    -   (iv) a gelation temperature between about 19° C. to about 42°        C.; and    -   (v) a mean dissolution time of about 30 hours for the auris        sensory cell modulating agent.

In some embodiments of the methods described above, the auris sensorycell modulating agent is released from the composition or device for aperiod of at least 3 days. In some embodiments of the methods describedabove, the auris sensory cell modulating agent is released from thecomposition or device for a period of at least 5 days. In someembodiments of the methods described above, the auris sensory cellmodulating agent is released from the composition or device for a periodof at least 10 days. In some embodiments of the method described above,the auris sensory cell modulating agent is essentially in the form ofmicronized particles.

In some embodiments of the methods described herein, the composition isadministered across the round window. In some embodiments, the oticand/or vestibular disorder is ototoxicity, chemotherapy-induced hearingloss, sensorineural hearing loss, noise induced hearing loss,excitotoxicity, Meniere's Disease/Syndrome, endolymphatic hydrops,labyrinthitis, Ramsay Hunt's Syndrome, vestibular neuronitis, tinnitusor microvascular compression syndrome.

Provided herein are pharmaceutical compositions or devices comprising anamount of an ototoxic agent that is therapeutically effective fortreating an otic disease or condition comprising substantially lowdegradation products of the ototoxic agent, the pharmaceuticalcomposition or device further comprising two or more characteristicsselected from:

-   -   (i) between about 0.1% to about 10% by weight of the ototoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) sterile water, q.s., buffered to provide a pH between        about 5.5 and about 8.0;    -   (iv) multiparticulate ototoxic agent;    -   (v) a gelation temperature between about 19° C. to about 42° C.;    -   (vi) less than about 50 colony forming units (cfu) of        microbiological agents per gram of formulation;    -   (vii) less than about 5 endotoxin units (EU) per kg of body        weight of a subject;    -   (viii) a mean dissolution time of about 30 hours for the        ototoxic agent; and    -   (ix) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments, the pharmaceutical composition or device comprises:

-   -   (i) between about 0.1% to about 10% by weight of the ototoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) multiparticulate ototoxic agent;    -   (iv) a gelation temperature between about 19° C. to about 42°        C.;    -   (v) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments, the pharmaceutical composition or device comprises:

-   -   (i) between about 0.1% to about 10% by weight of the ototoxic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) multiparticulate ototoxic agent;    -   (iv) a gelation temperature between about 19° C. to about 42°        C.;    -   (v) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments, the pharmaceutical composition or device comprisingan ototoxic agent provides a practical osmolarity between about 200 and400 mOsm/L. In some embodiments, the pharmaceutical composition ordevice comprising an ototoxic agent provides a practical osmolaritybetween about 250 and 320 mOsm/L.

In some embodiments, an ototoxic agent is released from the compositionor device for a period of at least 3 days. In some embodiments, anototoxic agent is released from the composition or device for a periodof at least 5 days. In some embodiments, the pharmaceutical compositionor device comprising an ototoxic agent is an auris-acceptablethermoreversible gel. In some embodiments, the pharmaceuticalcomposition or device comprises the ototoxic agent as essentially in theform of micronized particles.

Provided herein are pharmaceutical compositions or devices comprising anamount of a trophic agent that is therapeutically effective for treatingan otic disease or condition comprising substantially low degradationproducts of the trophic agent, the pharmaceutical composition or devicefurther comprising two or more characteristics selected from:

-   -   (i) between about 0.1% to about 10% by weight of the trophic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) sterile water, q.s., buffered to provide a pH between        about 5.5 and about 8.0;    -   (iv) multiparticulate trophic agent;    -   (v) a gelation temperature between about 19° C. to about 42° C.;    -   (vi) less than about 50 colony forming units (cfu) of        microbiological agents per gram of formulation;    -   (vii) less than about 5 endotoxin units (EU) per kg of body        weight of a subject;    -   (viii) a mean dissolution time of about 30 hours for the trophic        agent; and    -   (ix) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments, the pharmaceutical composition or device comprises:

-   -   (i) between about 0.1% to about 10% by weight of the trophic        agent, or pharmaceutically acceptable prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) multiparticulate trophic agent;    -   (iv) a gelation temperature between about 19° C. to about 42°        C.;    -   (viii) a mean dissolution time of about 30 hours for the trophic        agent; and    -   (ix) an apparent viscosity of about 100,000 cP to about 500,000        cP.

In some embodiments, the pharmaceutical composition or device comprisinga trophic agent provides a practical osmolarity between about 200 and400 mOsm/L. In some embodiments, the pharmaceutical composition ordevice comprising a trophic agent provides a practical osmolaritybetween about 250 and 320 mOsm/L. In some embodiments, the trophic agentis released from the composition or device for a period of at least 3days. In some embodiments, the trophic agent is released from thecomposition or device for a period of at least 5 days.

In some embodiments, the pharmaceutical composition or device comprisinga trophic agent is an auris-acceptable thermoreversible gel. In someembodiments, the pharmaceutical composition or device comprises thetrophic agent as essentially in the form of micronized particles.

In some embodiments any composition or device described above comprises:

-   -   (i) between about 0.1% to about 10% by weight of the auris        sensory cell modulating agent, or pharmaceutically acceptable        prodrug or salt thereof;    -   (ii) between about 14% to about 21% by weight of a        polyoxyethylene-polyoxypropylene triblock copolymer of general        formula E106 P70 E106;    -   (iii) multiparticulate auris sensory cell modulating agent;    -   (iv) a gelation temperature between about 19° C. to about 42°        C.; and    -   (v) a mean dissolution time of about 30 hours for the auris        sensory cell modulating agent.

In some embodiments, any pharmaceutical composition or device describedabove comprises substantially low degradation products of the aurissensory cell modulating agent.

In some embodiments a pharmaceutical composition or device describedabove provides a practical osmolarity between about 150 and 500 mOsm/L.In some embodiments a pharmaceutical composition or device describedabove provides a practical osmolarity between about 200 and 400 mOsm/L.In some embodiments a pharmaceutical composition or device describedabove provides a practical osmolarity between about 250 and 320 mOsm/L.

In some embodiments, the auris sensory cell modulating agent is releasedfrom the pharmaceutical composition or device described above for aperiod of at least 3 days. In some embodiments, the auris sensory cellmodulating agent is released from the pharmaceutical composition ordevice described above for a period of at least 5 days. In someembodiments, the auris sensory cell modulating agent is released fromthe pharmaceutical composition or device described above for a period ofat least 10 days. In some embodiments, the auris sensory cell modulatingagent is released from the pharmaceutical composition or devicedescribed above for a period of at least 14 days. In some embodiments,the auris sensory cell modulating agent is released from thepharmaceutical composition or device described above for a period of atleast one month.

In some embodiments, a pharmaceutical composition or device describedabove comprises auris sensory cell modulating agent as a neutralcompound, a free acid, a free base, a salt or a prodrug. In someembodiments, a pharmaceutical composition or device described abovecomprises auris sensory cell modulating agent as a neutral compound, afree acid, a free base, a salt or a prodrug, or a combination thereof.In some embodiments of the pharmaceutical compositions or devicesdescribed herein, the auris sensory cell modulating agent isadministered in the form of an ester prodrug.

In some embodiments, a pharmaceutical composition or device describedabove is an auris-acceptable thermoreversible gel. In some embodimentsof the pharmaceutical composition or device, thepolyoxyethylene-polyoxypropylene triblock copolymer is bioeliminated.

In some embodiments the pharmaceutical composition or device furthercomprises a penetration enhancer. In some embodiments, thepharmaceutical composition or device further comprises a dye.

In some embodiments, the pharmaceutical composition or device furthercomprises the auris sensory cell modulating agent, or pharmaceuticallyacceptable salt thereof, prodrug or combination thereof as an immediaterelease agent.

In some embodiments of the pharmaceutical composition or device theauris sensory cell modulating agent comprises multiparticulates. In someembodiments of the pharmaceutical composition or device the aurissensory cell modulating agent is essentially in the form of micronizedparticles. In some embodiments of the pharmaceutical composition ordevice, the auris sensory cell modulating agent is in the form ofmicronized auris sensory cell modulating agent powder.

In some embodiments, a pharmaceutical composition or device describedabove comprises about 10% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.In some embodiments, a pharmaceutical composition or device describedabove comprises about 15% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.In some embodiments, a pharmaceutical composition or device describedabove comprises about 20% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.In some embodiments, a pharmaceutical composition or device describedabove comprises about 25% of a polyoxyethylene-polyoxypropylene triblockcopolymer of general formula E106 P70 E106 by weight of the composition.

In some embodiments, a pharmaceutical composition or device describedabove comprises about 0.01% of an auris sensory cell modulating agent,or pharmaceutically acceptable prodrug or salt thereof, by weight of thecomposition. In some embodiments, a pharmaceutical composition or devicedescribed above comprises about 0.05% of an auris sensory cellmodulating agent, or pharmaceutically acceptable prodrug or saltthereof, by weight of the composition. In some embodiments, apharmaceutical composition or device described above comprises about0.1% of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the composition. Insome embodiments, a pharmaceutical composition or device described abovecomprises about 1% of an auris sensory cell modulating agent, orpharmaceutically acceptable prodrug or salt thereof, by weight of thecomposition. In some embodiments, a pharmaceutical composition or devicedescribed above comprises about 2.5% of an auris sensory cell modulatingagent, or pharmaceutically acceptable prodrug or salt thereof, by weightof the composition. In some embodiments, a pharmaceutical composition ordevice described above comprises about 5% of an auris sensory cellmodulating agent, or pharmaceutically acceptable prodrug or saltthereof, by weight of the composition. In some embodiments, apharmaceutical composition or device described above comprises about 10%of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the composition. Insome embodiments, a pharmaceutical composition or device described abovecomprises about 20% of an auris sensory cell modulating agent, orpharmaceutically acceptable prodrug or salt thereof, by weight of thecomposition. In some embodiments, a pharmaceutical composition or devicedescribed above comprises about 30% of an auris sensory cell modulatingagent, or pharmaceutically acceptable prodrug or salt thereof, by weightof the composition. In some embodiments, a pharmaceutical composition ordevice described above comprises about 40% of an auris sensory cellmodulating agent, or pharmaceutically acceptable prodrug or saltthereof, by weight of the composition. In some embodiments, apharmaceutical composition or device described above comprises up toabout 50% of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the composition.

In some embodiments, a pharmaceutical composition or device describedabove has a pH between about 5.5 to about 8.0. In some embodiments, apharmaceutical composition or device described above has a pH betweenabout 6.0 to about 8.0. In some embodiments, a pharmaceuticalcomposition or device described above has a pH between about 6.0 toabout 7.6. In some embodiments, a pharmaceutical composition or devicedescribed above has a pH between about 7.0 to about 7.6.

In some embodiments, a pharmaceutical composition or device describedabove contains less than 100 colony forming units (cfu) ofmicrobiological agents per gram of formulation. In some embodiments, apharmaceutical composition or device described above contains less than50 colony forming units (cfu) of microbiological agents per gram offormulation. In some embodiments, a pharmaceutical composition or devicedescribed above contains less than 10 colony forming units (cfu) ofmicrobiological agents per gram of formulation.

In some embodiments, a pharmaceutical composition or device describedabove contains less than 5 endotoxin units (EU) per kg of body weight ofa subject. In some embodiments, a pharmaceutical composition or devicedescribed above contains less than 4 endotoxin units (EU) per kg of bodyweight of a subject.

In some embodiments a pharmaceutical composition or device describedabove provides a gelation temperature between about between about 19° C.to about 42° C. In some embodiments a pharmaceutical composition ordevice described above provides a gelation temperature between aboutbetween about 19° C. to about 37° C. In some embodiments apharmaceutical composition or device described above provides a gelationtemperature between about between about 19° C. to about 30° C.

In some embodiments, the pharmaceutical composition or device is anauris-acceptable thermoreversible gel. In some embodiments, thepolyoxyethylene-polyoxypropylene triblock copolymer is biodegradableand/or bioeliminated (e.g., the copolymer is eliminated from the body bya biodegradation process, e.g., elimination in the urine, the feces orthe like). In some embodiments, a pharmaceutical composition or devicedescribed herein further comprises a mucoadhesive. In some embodiments,a pharmaceutical composition or device described herein furthercomprises a penetration enhancer. In some embodiments, a pharmaceuticalcomposition or device described herein further comprises a thickeningagent. In some embodiments, a pharmaceutical composition or devicedescribed herein further comprises a dye.

In some embodiments, a pharmaceutical composition or device describedherein further comprises a drug delivery device selected from a needleand syringe, a pump, a microinjection device, a wick, an in situ formingspongy material or combinations thereof.

In some embodiments, a pharmaceutical composition or device describedherein is a pharmaceutical composition or device wherein the aurissensory cell modulating agent, or pharmaceutically acceptable saltthereof, has limited or no systemic release, systemic toxicity, poor PKcharacteristics, or combinations thereof. In some embodimentspharmaceutical compositions or devices described herein comprise one ormore auris sensory cell modulating agent, or pharmaceutically acceptablesalt thereof, prodrug or combination thereof as an immediate releaseagent.

In some embodiments, pharmaceutical compositions or devices describedherein are pharmaceutical compositions or devices wherein the pH of thepharmaceutical composition or device is a perilymph-suitable pH betweenabout 6.0 to about 7.6.

In some embodiments of the pharmaceutical compositions or devicesdescribed herein, the ratio of a polyoxyethylene-polyoxypropylenetriblock copolymer of general formula E106 P70 E106 to a thickeningagent is from about 40:1 to about 5:1. In some embodiments, thethickening agent is carboxymethyl cellulose, hydroxypropyl cellulose orhydroxypropyl methylcellulose.

In some embodiments, the otic and/or vestibular disorder is ototoxicity,chemotherapy-induced hearing loss, sensorineural hearing loss, noiseinduced hearing loss, excitotoxicity, Meniere's Disease/Syndrome,endolymphatic hydrops, labyrinthitis, Ramsay Hunt's Syndrome, vestibularneuronitis, tinnitus or microvascular compression syndrome.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. illustrates a comparison of non-sustained release and sustainedrelease formulations.

FIG. 2 illustrates the effect of concentration on the viscosity ofaqueous solutions of Blanose refined CMC.

FIG. 3 illustrates the effect of concentration on the viscosity ofaqueous solutions of Methocel.

FIG. 4 illustrates the anatomy of the ear

FIG. 5 shows predicted tunable release characteristics from fourcompositions

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are controlled release compositions for treating orameliorating hearing loss or reduction resulting from destroyed,stunted, malfunctioning, damaged, fragile or missing hairs in the innerear. In one embodiment, the controlled release composition comprises atherapeutically-effective amount of at least one modulator of growthand/or regeneration of auris sensory cells (e.g., neuron and/or haircells of the auris), a controlled release auris-acceptable excipient,and an auris-acceptable vehicle. In one embodiment, the controlledrelease composition comprises a therapeutically-effective amount of atleast one modulator of auris sensory cell damage, a controlled releaseauris-acceptable excipient, and an auris-acceptable vehicle. In oneembodiment, the controlled release composition comprises atherapeutically-effective amount of at least one agent that protectsneuron and/or hair cells of the auris from damage or reduces, reversesor delays damage to auris sensory cells, a controlled releaseauris-acceptable excipient, and an auris-acceptable vehicle.

Further disclosed herein are controlled release auris sensory cellmodulating agent compositions and formulations to treat ototoxicity,excitotoxicity, sensorineural hearing loss, noise induced hearing loss,Meniere's Disease/Syndrome, endolymphatic hydrops, labyrinthitis, RamsayHunt's Syndrome, vestibular neuronitis, tinnitus and microvascularcompression syndrome.

A few therapeutic products are available for the treatment ofototoxicity, excitotoxicity, sensorineural hearing loss, noise inducedhearing loss, Meniere's Disease/Syndrome, endolymphatic hydrops,labyrinthitis, Ramsay Hunt's Syndrome, vestibular neuronitis, tinnitusand microvascular compression syndrome; however, systemic routes viaoral, intravenous or intramuscular routes are currently used to deliverthese therapeutic agents.

Systemic drug administration may create a potential inequality in drugconcentration with higher circulating levels in the serum, and lowerlevels in the target auris interna organ structures. As a result, fairlylarge amounts of drug are required to overcome this inequality in orderto deliver sufficient, therapeutically effective quantities to the innerear. Further, bioavailability is often decreased due to metabolism ofthe drug by the liver. In addition, systemic drug administration mayincrease the likelihood of systemic toxicities and adverse side effectsas a result of the high serum amounts required to effectuate sufficientlocal delivery to the target site. Systemic toxicities may also occur asa result of liver breakdown and processing of the therapeutic agents,forming toxic metabolites that effectively erase any benefit attainedfrom the administered therapeutic (e.g. metabolites of carbamazepine cancause hepatic injuries and death in some patients).

To overcome the toxic and attendant undesired side effects of systemicdelivery of auris sensory cell modulating agents (which are generallyunderstood to be toxic to cells), disclosed herein are methods andcompositions for local delivery of auris sensory cell modulating agentsto auris media and/or auris interna structures. Access to, for example,the vestibular and cochlear apparatus will occur through the auris mediaor auris interna, including the round window membrane, the ovalwindow/stapes footplate, the annular ligament and through the oticcapsule/temporal bone. In further or alternative embodiments, the auriscontrolled-release formulations are capable of being administered on ornear the round window membrane via intratympanic injection. In otherembodiments, the auris controlled release formulations are administeredon or near the round window or the crista fenestrae cochleae throughentry via a post-auricular incision and surgical manipulation into ornear the round window or the crista fenestrae cochleae area.Alternatively, the auris controlled release formulation is applied viasyringe and needle, wherein the needle is inserted through the tympanicmembrane and guided to the area of the round window or crista fenestraecochleae.

In addition, localized treatment of the auris interna also affords theuse of previously undesired therapeutic agents, including agents withpoor pK profiles, poor uptake, low systemic release, and/or toxicityissues.

Because of the localized targeting of the auris sensory cell modulatingagent formulations and compositions, as well as the biological bloodbarrier present in the auris interna, the risk of adverse effects willbe reduced as a result of treatment with previously characterized toxicor ineffective auris sensory cell modulating agent. Accordingly, alsocontemplated within the scope of the embodiments herein is the use ofauris sensory cell modulating agents in the treatment of ototoxicity,excitotoxicity, sensorineural hearing loss, noise induced hearing loss,Meniere's Disease/Syndrome, endolymphatic hydrops, labyrinthitis, RamsayHunt's Syndrome, vestibular neuronitis, tinnitus and microvascularcompression syndrome, including therapeutic agents that have beenpreviously rejected by practitioners because of adverse effects orineffectiveness of the auris sensory cell modulating agent(s).

Also included within the embodiments disclosed herein is the use ofadditional auris media and/or auris interna-acceptable agents incombination with the auris sensory cell modulating agent formulationsand compositions disclosed herein. When used, such agents assist in thetreatment of hearing or equilibrium loss or dysfunction resulting froman autoimmune disorder, including vertigo, tinnitus, hearing loss,balance disorders, infections, inflammatory response or combinationsthereof. Accordingly, agents that ameliorate or reduce the effects ofvertigo, tinnitus, hearing loss, balance disorders, infections,inflammatory response or combinations thereof are also contemplated tobe used in combination with the auris sensory cell modulating agent(s)described herein.

In some embodiments, the composition further comprises an auris sensorycell modulating agent as an immediate release agent wherein theimmediate release auris sensory cell modulating agent is the same agentas the controlled-release agent, a different auris sensory cellmodulating agent, an additional therapeutic agent, or a combinationthereof. In some embodiments, the composition further comprises anadditional therapeutic agent. In some embodiments, the additionaltherapeutic agent is an anesthetic, a local acting anesthetic, ananalgesic, an antibiotic, an antiemetic, an antifungal, ananti-microbial agent, an antiseptic, an antiviral, a chemotherapeuticagent, a diuretic, a keratolytic agent, an otoprotectant, animmunosuppressant comprising but not limited to such agents ascalcineurin inhibitors (cyclosporine, tacrolimus, pimecrolimus),macrolides such as rapamycin, corticosteroids or combinations thereof.In some embodiments, the additional therapeutic agent is an immediaterelease agent. In some embodiments, the additional therapeutic agent isa controlled release agent.

Accordingly, provided herein are controlled release auris sensory cellmodulating agent formulations and compositions to locally treat aurismedia and/or auris interna structures, thereby avoiding side effects asa result of systemic administration of the auris sensory cell modulatingagents. The locally applied auris sensory cell modulating agentformulations and compositions are compatible with auris media and/orauris interna structures, and are administered either directly to thedesired auris media and/or auris interna structure, e.g. the cochlearregion or the tympanic cavity, or administered to a structure in directcommunication with areas of the auris interna, including but not limitedto the round window membrane, the crista fenestrae cochleae or the ovalwindow membrane. By specifically targeting the auris media or aurisinterna structures, adverse side effects as a result of systemictreatment are avoided. Moreover, by providing a controlled release aurissensory cell modulating agent formulation or composition to treat oticdisorders, a constant and/or extended source of auris sensory cellmodulating agent is provided to the individual or patient suffering froman otic disorder, reducing or eliminating the variability of treatment.

Intratympanic injection of therapeutic agents is the technique ofinjecting a therapeutic agent behind the tympanic membrane into theauris media and/or auris interna. Despite early success with thistechnique (Schuknecht, Laryngoscope (1956) 66, 859-870) some challengesdo remain. For example, access to the round window membrane, the site ofdrug absorption into the auris interna, can be challenging.

However, intra-tympanic injections create several unrecognized problemsnot addressed by currently available treatment regimens, such aschanging the osmolarity and pH of the perilymph and endolymph, andintroducing pathogens and endotoxins that directly or indirectly damageinner ear structures. One of the reasons the art may not have recognizedthese problems is that there are no approved intra-tympaniccompositions: the inner ear provides sui generis formulation challenges.Thus, compositions developed for other parts of the body have little tono relevance for an intra-tympanic composition.

There is no guidance in the prior art regarding requirements (e.g.,level of sterility, pH, osmolarity) for otic formulations that aresuitable for administration to humans. There is wide anatomicaldisparity between the ears of animals across species. A consequence ofthe inter-species differences in auditory structures is that animalmodels of inner ear disease are often unreliable as a tool for testingtherapeutics that are being developed for clinical approval.

Provided herein are otic formulations that meet stringent criteria forpH, osmolarity, ionic balance, sterility, endotoxin and/or pyrogenlevels. The auris compositions described herein are compatible with themicroenvironment of the inner ear (e.g., the perilymph) and are suitablefor administration to humans. In some embodiments, the formulationsdescribed herein comprise dyes and aid visualization of the administeredcompositions obviating the need for invasive procedures (e.g., removalof perilymph) during preclinical and/or clinical development ofintratympanic therapeutics.

Provided herein are controlled release auris sensory cell modulatingagent formulations and compositions to locally treat targeted aurisstructures, thereby avoiding side effects as a result of systemicadministration of the auris sensory cell modulating agent formulationsand compositions. The locally applied auris sensory cell modulatingagent formulations and compositions and devices are compatible with thetargeted auris structures, and administered either directly to thedesired targeted auris structure, e.g. the cochlear region, the tympaniccavity or the external ear, or administered to a structure in directcommunication with areas of the auris interna, including but not limitedto the round window membrane, the crista fenestrae cochleae or the ovalwindow membrane. By specifically targeting an auris structure, adverseside effects as a result of systemic treatment are avoided. Moreover,clinical studies have shown the benefit of having long term exposure ofdrug to the perilymph of the cochlea, for example with improved clinicalefficacy of sudden hearing loss when the therapeutic agent is given onmultiple occasions. Thus, by providing a controlled release aurissensory cell modulating agent formulation or composition to treat oticdisorders, a constant, and/or extended source of auris sensory cellmodulating agent is provided to the individual or patient suffering froman otic disorder, reducing or eliminating variabilities in treatment.Accordingly, one embodiment disclosed herein is to provide a compositionthat enables at least one auris sensory cell modulating agent to bereleased in therapeutically effective doses either at variable orconstant rates such as to ensure a continuous release of the at leastone agent. In some embodiments, the auris sensory cell modulating agentsdisclosed herein are administered as an immediate release formulation orcomposition. In other embodiments, the auris sensory cell modulatingagents are administered as a sustained release formulation, releasedeither continuously, variably or in a pulsatile manner, or variantsthereof. In still other embodiments, auris sensory cell modulating agentformulation is administered as both an immediate release and sustainedrelease formulation, released either continuously, variably or in apulsatile manner, or variants thereof. The release is optionallydependent on environmental or physiological conditions, for example, theexternal ionic environment (see, e.g. Oros® release system, Johnson &Johnson).

In addition, the auris-acceptable controlled-release auris sensory cellmodulating agent formulations and treatments described herein areprovided to the target ear region of the individual in need, includingthe inner ear, and the individual in need is additionally administeredan oral dose of auris sensory cell modulating agent. In someembodiments, the oral dose of auris sensory cell modulating agent isadministered prior to administration of the auris-acceptablecontrolled-release auris sensory cell modulating agent formulation, andthen the oral dose is tapered off over the period of time that theauris-acceptable controlled-release auris sensory cell modulating agentformulation is provided. Alternatively, the oral dose of auris sensorycell modulating agent is administered during administration of theauris-acceptable controlled-release auris sensory cell modulating agentformulation, and then the oral dose is tapered off over the period oftime that the auris-acceptable controlled-release auris sensory cellmodulating agent formulation is provided. Alternatively, the oral doseof auris sensory cell modulating agent is administered afteradministration of the auris-acceptable controlled-release auris sensorycell modulating agent formulation has been initiated, and then the oraldose is tapered off over the period of time that the auris-acceptablecontrolled-release auris sensory cell modulating agent formulation isprovided.

In addition, the auris sensory cell modulating agent pharmaceuticalcompositions or formulations or devices included herein also includecarriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. Such carriers, adjuvants, and other excipientswill be compatible with the environment in the targeted aurisstructure(s). Accordingly, specifically contemplated are carriers,adjuvants and excipients that lack ototoxicity or are minimally ototoxicin order to allow effective treatment of the otic disorders contemplatedherein with minimal side effects in the targeted regions or areas. Toprevent ototoxicity, auris sensory cell modulating agent pharmaceuticalcompositions or formulations or devices disclosed herein are optionallytargeted to distinct regions of the targeted auris structures, includingbut not limited to the tympanic cavity, vestibular bony and membranouslabyrinths, cochlear bony and membranous labyrinths and other anatomicalor physiological structures located within the auris interna.

Certain Definitions

The term “auris-acceptable” with respect to a formulation, compositionor ingredient, as used herein, includes having no persistent detrimentaleffect on the auris interna (or inner ear) of the subject being treated.By “auris-pharmaceutically acceptable,” as used herein, refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound in reference to theauris interna (or inner ear), and is relatively or is reduced intoxicity to the auris interna (or inner ear), i.e., the material isadministered to an individual without causing undesirable biologicaleffects or interacting in a deleterious manner with any of thecomponents of the composition in which it is contained.

As used herein, amelioration or lessening of the symptoms of aparticular otic disease, disorder or condition by administration of aparticular compound or pharmaceutical composition refers to any decreaseof severity, delay in onset, slowing of progression, or shortening ofduration, whether permanent or temporary, lasting or transient that isattributed to or associated with administration of the compound orcomposition.

“Antioxidants” are auris-pharmaceutically acceptable antioxidants, andinclude, for example, butylated hydroxytoluene (BHT), sodium ascorbate,ascorbic acid, sodium metabisulfite and tocopherol. In certainembodiments, antioxidants enhance chemical stability where required.Antioxidants are also used to counteract the ototoxic effects of certaintherapeutic agents, including agents that are used in combination withthe auris sensory cell modulating agents disclosed herein.

“Auris interna” refers to the inner ear, including the cochlea and thevestibular labyrinth, and the round window that connects the cochleawith the middle ear.

“Auris-interna bioavailability” refers to the percentage of theadministered dose of compounds disclosed herein that becomes availablein the inner ear of the animal or human being studied.

“Auris media” refers to the middle ear, including the tympanic cavity,auditory ossicles and oval window, which connects the middle ear withthe inner ear.

“Balance disorder” refers to a disorder, illness, or condition whichcauses a subject to feel unsteady, or to have a sensation of movement.Included in this definition are dizziness, vertigo, disequilibrium, andpre-syncope. Diseases which are classified as balance disorders include,but are not limited to, Ramsay Hunt's Syndrome, Meniere's Disease, malde debarquement, benign paroxysmal positional vertigo, andlabyrinthitis.

“Blood plasma concentration” refers to the concentration of compoundsprovided herein in the plasma component of blood of a subject.

“Carrier materials” are excipients that are compatible with the aurissensory cell modulating agent, the auris interna and the release profileproperties of the auris-acceptable pharmaceutical formulations. Suchcarrier materials include, e.g., binders, suspending agents,disintegration agents, filling agents, surfactants, solubilizers,stabilizers, lubricants, wetting agents, diluents, and the like.“Auris-pharmaceutically compatible carrier materials” include, but arenot limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like.

The term “diluent” refers to chemical compounds that are used to dilutethe auris sensory cell modulating agent prior to delivery and which arecompatible with the auris interna.

“Dispersing agents,” and/or “viscosity modulating agents” are materialsthat control the diffusion and homogeneity of the auris sensory cellmodulating agent through liquid media. Examples of diffusionfacilitators/dispersing agents include but are not limited tohydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolhas a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose are also be used as dispersing agents. Dispersingagents useful in liposomal dispersions and self-emulsifying dispersionsof the auris sensory cell modulating agents disclosed herein aredimyristoyl phosphatidyl choline, natural phosphatidyl choline fromeggs, natural phosphatidyl glycerol from eggs, cholesterol and isopropylmyristate.

“Drug absorption” or “absorption” refers to the process of movement ofthe auris sensory cell modulating agents from the localized site ofadministration, by way of example only, the round window membrane of theinner ear, and across a barrier (the round window membranes, asdescribed below) into the auris interna or inner ear structures. Theterms “co-administration” or the like, as used herein, are meant toencompass administration of the auris sensory cell modulating agents toa single patient, and are intended to include treatment regimens inwhich the auris sensory cell modulating agents are administered by thesame or different route of administration or at the same or differenttime.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of the auris sensory cellmodulating agent being administered that would be expected to relieve tosome extent one or more of the symptoms of the disease or conditionbeing treated. For example, the result of administration of the aurissensory cell modulating agent disclosed herein is reduction and/oralleviation of the signs, symptoms, or causes of tinnitus or balancedisorders. For example, an “effective amount” for therapeutic uses isthe amount of auris sensory cell modulating agent, including aformulation as disclosed herein required to provide a decrease oramelioration in disease symptoms without undue adverse side effects. Theterm “therapeutically effective amount” includes, for example, aprophylactically effective amount. An “effective amount” of a modulatorof neuron and/or hair cells of the auris composition disclosed herein isan amount effective to achieve a desired pharmacologic effect ortherapeutic improvement without undue adverse side effects. It isunderstood that “an effective amount” or “a therapeutically effectiveamount” varies, in some embodiments, from subject to subject, due tovariation in metabolism of the compound administered, age, weight,general condition of the subject, the condition being treated, theseverity of the condition being treated, and the judgment of theprescribing physician. It is also understood that “an effective amount”in an extended-release dosing format may differ from “an effectiveamount” in an immediate release dosign format based upon pharmacokineticand pharmacodynamic considerations.

The terms “enhance” or “enhancing” refers to an increase or prolongationof either the potency or duration of a desired effect of auris sensorycell modulating agent, or a diminution of any adverse symptomatologythat is consequent upon the administration of the therapeutic agent.Thus, in regard to enhancing the effect of the auris sensory cellmodulating agents disclosed herein, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents that are used in combination with theauris sensory cell modulating agent disclosed herein. An“enhancing-effective amount,” as used herein, refers to an amount ofauris sensory cell modulating agent or other therapeutic agent which isadequate to enhance the effect of another therapeutic agent or aurissensory cell modulating agent of the target auris structure in a desiredsystem. When used in a patient, amounts effective for this use willdepend on the severity and course of the disease, disorder or condition,previous therapy, the patient's health status and response to the drugs,and the judgment of the treating physician.

The term “inhibiting” includes preventing, slowing, or reversing thedevelopment of a condition, for example, or advancement of a conditionin a patient necessitating treatment.

The terms “kit” and “article of manufacture” are used as synonyms.

“Pharmacodynamics” refers to the factors which determine the biologicresponse observed relative to the concentration of drug at the desiredsite within the auris media and/or auris interna.

“Pharmacokinetics” refers to the factors which determine the attainmentand maintenance of the appropriate concentration of drug at the desiredsite within the auris media and/or auris interna.

“Modulator of neuron and/or hair cells of the auris” and “auris sensorycell modulating agent” are synonyms. They include agents that promotethe growth and/or regeneration of neurons and/or the hair cells of theauris, and agents that destroy neurons and/or hair cells of the auris.In some embodiments, an auris sensory cell modulating agent providestherapeutic benefit (e.g., alleviation of hearing loss) by promoting thegrowth and/or regeneration of auris sensory cells (e.g., neurons and/orthe hair cells) of the auris. In some embodiments, an auris sensory cellmodulating agent (e.g., a toxicant) provides therapeutic benefit (e.g.,alleviation of vertigo) by destroying or damaging auris sensory cells(e.g., neurons and/or hair cells of the auris). In some embodiments, anauris sensory cell modulating agent provides therapeutic benefit (e.g.,alleviation of tinnitus due to acoustic trauma) by treating and/orreversing damage to auris sensory cells (e.g., dysfunction of neuronsand/or hair cells of the auris) or reducing or delaying further damage(e.g., cell death) to auris sensory cells (e.g., by exerting anotoprotectant effect or a trophic effect).

The term “trophic agent” means an agent that promotes the survival,growth and/or regeneration of auris sensory cells (e.g., neurons and/orthe hair cells of the auris). In some embodiments, a trophic agentreduces or inhibits oxidative damage and/or osteoneogenesis and/ordegeneration of auris sensory cells. In some embodiments, a trophicagent maintains healthy auris sensory cells (e.g., after a surgicalimplant of a medical device). In some embodiments, a trophic agentupregulates activity of antioxidant enzymes (e.g., during administrationof ototoxic agents). In some embodiments, a trophic agent is animmunosuppressant (e.g., an immunosuppressant used during otic surgery).In some embodiments, a trophic agent is a growth factor (e.g., a growthfactor used after an implantation procedure to promote growth of auriscells).

The term “glutamate receptor antagonist” means a compound thatinterferes with or inhibits the activity of a glutamate receptor. Insome embodiments, the receptor is an AMPA receptor, or NMDA receptor. Insome embodiments, a glutamate receptor antagonist binds to a glutamatereceptor but said binding does not produce a physiological response.Glutamate receptor antagonists include partial agonists, inverseagonists, neutral or competitive antagonists, non-competitiveantagonists, allosteric antagonists, and/or orthosteric antagonists.

The term “glutamate receptor agonist” means a compound that binds to aglutamate receptor and activates the receptor. The term further includesa compound that facilitates the binding of a native ligand. In someembodiments, the receptor is an mGlu receptor. Glutamate receptoragonists include partial antagonists, allosteric agonists, and/ororthosteric agonists.

In prophylactic applications, compositions comprising the auris sensorycell modulating agents described herein are administered to a patientsusceptible to or otherwise at risk of a particular disease, disorder orcondition. For example, such conditions include and are not limited toototoxicity, excitotoxicity, sensorineural hearing loss, noise inducedhearing loss, Meniere's Disease/Syndrome, endolymphatic hydrops,labyrinthitis, Ramsay Hunt's Syndrome, vestibular neuronitis, tinnitusand microvascular compression syndrome. Such an amount is defined to bea “prophylactically effective amount or dose.” In this use, the preciseamounts also depend on the patient's state of health, weight, and thelike.

As used herein, a “pharmaceutical device” includes any compositiondescribed herein that, upon administration to an ear, provides areservoir for extended release of an active agent described herein.

The term “substantially low degradation products” means less than 5% byweight of the active agent are degradation products of the active agent.In further embodiments, the term means less than 3% by weight of theactive agent are degradation products of the active agent. In yetfurther embodiments, the term means less than 2% by weight of the activeagent are degradation products of the active agent. In furtherembodiments, the term means less than 1% by weight of the active agentare degradation products of the active agent. In some embodiments, anyindividual impurity (e.g., metal impurity, degradation products ofactive agent and/or excipients, or the like) present in a formulationdescribed herein is less than 5%, less than 2%, or less than 1% byweight of the active agent. In some embodiments the formulation does notcontain precipitate during storage or change in color aftermanufacturing and storage.

As used herein “essentially in the form of micronized powder” includes,by way of example only, greater than 70% by weight of the active agentis in the form of micronized particles of the active agent. In furtherembodiments, the term means greater than 80% by weight of the activeagent is in the form of micronized particles of the active agent. In yetfurther embodiments, the term means greater than 90% by weight of theactive agent is in the form of micronized particles of the active agent.

The term “otic intervention” means an external insult or trauma to oneor more auris structures and includes implants, otic surgery,injections, cannulations, or the like. Implants include auris-interna orauris-media medical devices, examples of which include cochlearimplants, hearing sparing devices, hearing-improvement devices, shortelectrodes, micro-prostheses or piston-like prostheses; needles; stemcell transplants; drug delivery devices; any cell-based therapeutic; orthe like. Otic surgery includes middle ear surgery, inner ear surgery,tympanostomy, cochleostomy, labyrinthotomy, mastoidectomy, stapedectomy,stapedotomy, endolymphatic sacculotomy or the like. Injections includeintratympanic injections, intracochlear injections, injections acrossthe round window membrane or the like. Cannulations includeintratympanic, intracochlear, endolymphatic, perilymphatic or vestibularcannulations or the like.

A “prodrug” refers to an auris sensory cell modulating agent that isconverted into the parent drug in vivo. In certain embodiments, aprodrug is enzymatically metabolized by one or more steps or processesto the biologically, pharmaceutically or therapeutically active form ofthe compound. To produce a prodrug, a pharmaceutically active compoundis modified such that the active compound will be regenerated upon invivo administration. In one embodiment, the prodrug is designed to alterthe metabolic stability or the transport characteristics of a drug, tomask side effects or toxicity, or to alter other characteristics orproperties of a drug. Compounds provided herein, in some embodiments,are derivatized into suitable prodrugs.

“Solubilizers” refer to auris-acceptable compounds such as triacetin,triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate,sodium doccusate, vitamin E TPGS, dimethylacetamide,N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone,hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol,n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethyleneglycol 200-600, glycofurol, transcutol, propylene glycol, and dimethylisosorbide and the like that assist or increase the solubility of theauris sensory cell modulating agents disclosed herein.

“Stabilizers” refers to compounds such as any antioxidation agents,buffers, acids, preservatives and the like that are compatible with theenvironment of the auris interna. Stabilizers include but are notlimited to agents that will do any of (1) improve the compatibility ofexcipients with a container, or a delivery system, including a syringeor a glass bottle, (2) improve the stability of a component of thecomposition, or (3) improve formulation stability.

“Steady state,” as used herein, is when the amount of drug administeredto the auris interna is equal to the amount of drug eliminated withinone dosing interval resulting in a plateau or constant levels of drugexposure within the targeted structure.

As used herein, the term “subject” is used to mean an animal, preferablya mammal, including a human or non-human. The terms patient and subjectmay be used interchangeably.

“Surfactants” refer to compounds that are auris-acceptable, such assodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin,vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitanmonooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate,copolymers of ethylene oxide and propylene oxide, e.g., Pluronic®(BASF), and the like. Some other surfactants include polyoxyethylenefatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60)hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenylethers, e.g., octoxynol 10, octoxynol 40. In some embodiments,surfactants are included to enhance physical stability or for otherpurposes.

The terms “treat,” “treating” or “treatment,” as used herein, includealleviating, abating or ameliorating a disease or condition, for exampletinnitus, symptoms, preventing additional symptoms, ameliorating orpreventing the underlying metabolic causes of symptoms, inhibiting thedisease or condition, e.g., arresting the development of the disease orcondition, relieving the disease or condition, causing regression of thedisease or condition, relieving a condition caused by the disease orcondition, or stopping the symptoms of the disease or condition eitherprophylactically and/or therapeutically.

Other objects, features, and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only.

Anatomy of the Ear

As shown in FIG. 4, the outer ear is the external portion of the organand is composed of the pinna (auricle), the auditory canal (externalauditory meatus) and the outward facing portion of the tympanicmembrane, also known as the ear drum. The pinna, which is the fleshypart of the external ear that is visible on the side of the head,collects sound waves and directs them toward the auditory canal. Thus,the function of the outer ear, in part, is to collect and direct soundwaves towards the tympanic membrane and the middle ear.

The middle ear is an air-filled cavity, called the tympanic cavity,behind the tympanic membrane. The tympanic membrane, also known as theear drum, is a thin membrane that separates the external ear from themiddle ear. The middle ear lies within the temporal bone, and includeswithin this space the three ear bones (auditory ossicles): the malleus,the incus and the stapes. The auditory ossicles are linked together viatiny ligaments, which form a bridge across the space of the tympaniccavity. The malleus, which is attached to the tympanic membrane at oneend, is linked to the incus at its anterior end, which in turn is linkedto the stapes. The stapes is attached to the oval window, one of twowindows located within the tympanic cavity. A fibrous tissue layer,known as the annular ligament connects the stapes to the oval window.Sound waves from the outer ear first cause the tympanic membrane tovibrate. The vibration is transmitted across to the cochlea through theauditory ossicles and oval window, which transfers the motion to thefluids in the auris interna. Thus, the auditory ossicles are arranged toprovide a mechanical linkage between the tympanic membrane and the ovalwindow of the fluid-filled auris interna, where sound is transformed andtransduced to the auris interna for further processing. Stiffness,rigidity or loss of movement of the auditory ossicles, tympanic membraneor oval window leads to hearing loss, e.g. otosclerosis, or rigidity ofthe stapes bone.

The tympanic cavity also connects to the throat via the eustachian tube.The eustachian tube provides the ability to equalize the pressurebetween the outside air and the middle ear cavity. The round window, acomponent of the auris interna but which is also accessible within thetympanic cavity, opens into the cochlea of the auris interna. The roundwindow is covered by round window membrane, which consists of threelayers: an external or mucous layer, an intermediate or fibrous layer,and an internal membrane, which communicates directly with the cochlearfluid. The round window, therefore, has direct communication with theauris interna via the internal membrane.

Movements in the oval and round window are interconnected, i.e. as thestapes bone transmits movement from the tympanic membrane to the ovalwindow to move inward against the auris interna fluid, the round window(round window membrane) is correspondingly pushed out and away from thecochlear fluid. This movement of the round window allows movement offluid within the cochlea, which leads in turn to movement of thecochlear inner hair cells, allowing hearing signals to be transduced.Stiffness and rigidity in round window membrane leads to hearing lossbecause of the lack of ability of movement in the cochlear fluid. Recentstudies have focused on implanting mechanical transducers onto the roundwindow, which bypasses the normal conductive pathway through the ovalwindow and provides amplified input into the cochlear chamber.

Auditory signal transduction takes place in the auris interna. Thefluid-filled auris interna, or inner ear, consists of two majorcomponents: the cochlear and the vestibular apparatus. The auris internais located in part within the osseous or bony labyrinth, an intricateseries of passages in the temporal bone of the skull. The vestibularapparatus is the organ of balance and consists of the threesemi-circular canals and the vestibule. The three semi-circular canalsare arranged relative to each other such that movement of the head alongthe three orthogonal planes in space can be detected by the movement ofthe fluid and subsequent signal processing by the sensory organs of thesemi-circular canals, called the crista ampullaris. The cristaampullaris contains hair cells and supporting cells, and is covered by adome-shaped gelatinous mass called the cupula. The hairs of the haircells are embedded in the cupula. The semi-circular canals detectdynamic equilibrium, the equilibrium of rotational or angular movements.

When the head turns rapidly, the semicircular canals move with the head,but endolymph fluid located in the membranous semi-circular canals tendsto remain stationary. The endolymph fluid pushes against the cupula,which tilts to one side. As the cupula tilts, it bends some of the hairson the hair cells of the crista ampullaris, which triggers a sensoryimpulse. Because each semicircular canal is located in a differentplane, the corresponding crista ampullaris of each semi-circular canalresponds differently to the same movement of the head. This creates amosaic of impulses that are transmitted to the central nervous system onthe vestibular branch of the vestibulocochlear nerve. The centralnervous system interprets this information and initiates the appropriateresponses to maintain balance. Of importance in the central nervoussystem is the cerebellum, which mediates the sense of balance andequilibrium.

The vestibule is the central portion of the auris interna and containsmechanoreceptors bearing hair cells that ascertain static equilibrium,or the position of the head relative to gravity. Static equilibriumplays a role when the head is motionless or moving in a straight line.The membranous labyrinth in the vestibule is divided into two sac-likestructures, the utricle and the saccule. Each structure in turn containsa small structure called a macula, which is responsible for maintenanceof static equilibrium. The macula consists of sensory hair cells, whichare embedded in a gelatinous mass (similar to the cupula) that coversthe macula. Grains of calcium carbonate, called otoliths, are embeddedon the surface of the gelatinous layer.

When the head is in an upright position, the hairs are straight alongthe macula. When the head tilts, the gelatinous mass and otoliths tiltscorrespondingly, bending some of the hairs on the hair cells of themacula. This bending action initiates a signal impulse to the centralnervous system, which travels via the vestibular branch of thevestibulocochlear nerve, which in turn relays motor impulses to theappropriate muscles to maintain balance.

The cochlea is the portion of the auris interna related to hearing. Thecochlea is a tapered tube-like structure which is coiled into a shaperesembling a snail. The inside of the cochlea is divided into threeregions, which is further defined by the position of the vestibularmembrane and the basilar membrane. The portion above the vestibularmembrane is the scala vestibuli, which extends from the oval window tothe apex of the cochlea and contains perilymph fluid, an aqueous liquidlow in potassium and high in sodium content. The basilar membranedefines the scala tympani region, which extends from the apex of thecochlea to the round window and also contains perilymph. The basilarmembrane contains thousands of stiff fibers, which gradually increase inlength from the round window to the apex of the cochlea. The fibers ofthe basement membrane vibrate when activated by sound. In between thescala vestibuli and the scala tympani is the cochlear duct, which endsas a closed sac at the apex of the cochlea. The cochlear duct containsendolymph fluid, which is similar to cerebrospinal fluid and is high inpotassium.

The organ of Corti, the sensory organ for hearing, is located on thebasilar membrane and extends upward into the cochlear duct. The organ ofCorti contains hair cells, which have hairlike projections that extendfrom their free surface, and contacts a gelatinous surface called thetectorial membrane. Although hair cells have no axons, they aresurrounded by sensory nerve fibers that form the cochlear branch of thevestibulocochlear nerve (cranial nerve VIII).

As discussed, the oval window, also known as the elliptical windowcommunicates with the stapes to relay sound waves that vibrate from thetympanic membrane. Vibrations transferred to the oval window increasespressure inside the fluid-filled cochlea via the perilymph and scalavestibuli/scala tympani, which in turn causes the round window membraneto expand in response. The concerted inward pressing of the ovalwindow/outward expansion of the round window allows for the movement offluid within the cochlea without a change of intra-cochlear pressure.However, as vibrations travel through the perilymph in the scalavestibuli, they create corresponding oscillations in the vestibularmembrane. These corresponding oscillations travel through the endolymphof the cochlear duct, and transfer to the basilar membrane. When thebasilar membrane oscillates, or moves up and down, the organ of Cortimoves along with it. The hair cell receptors in the Organ of Corti thenmove against the tectorial membrane, causing a mechanical deformation inthe tectorial membrane. This mechanical deformation initiates the nerveimpulse which travels via the vestibulocochlear nerve to the centralnervous system, mechanically transmitting the sound wave received intosignals that are subsequently processed by the central nervous system.

Diseases

Otic disorders produce symptoms which include but are not limited tohearing loss, nystagmus, vertigo, tinnitus, inflammation, infection andcongestion. The otic disorders which are treated with the compositionsdisclosed herein are numerous and include ototoxicity, excitotoxicity,sensorineural hearing loss, noise induced hearing loss, Meniere'sDisease/Syndrome, endolymphatic hydrops, labyrinthitis, Ramsay Hunt'sSyndrome, vestibular neuronitis, tinnitus and microvascular compressionsyndrome.

Excitotoxicity

Excitotoxicity refers to the death or damaging of neurons and/or otichair cells by glutamate and/or similar substances.

Glutamate is the most abundant excitatory neurotransmitter in thecentral nervous system. Pre-synaptic neurons release glutamate uponstimulation. It flows across the synapse, binds to receptors located onpost-synaptic neurons, and activates these neurons. The glutamatereceptors include the NMDA, AMPA, and kainate receptors. Glutamatetransporters are tasked with removing extracellular glutamate from thesynapse. Certain events (e.g. ischemia or stroke) can damage thetransporters. This results in excess glutamate accumulating in thesynapse. Excess glutamate in synapses results in the over-activation ofthe glutamate receptors.

The AMPA receptor is activated by the binding of both glutamate andAMPA. Activation of certain isoforms of the AMPA receptor results in theopening of ion channels located in the plasma membrane of the neuron.When the channels open, Na+ and Ca2+ ions flow into the neuron and K+ions flow out of the neuron.

The NMDA receptor is activated by the binding of both glutamate andNMDA. Activation of the NMDA receptor, results in the opening of ionchannels located in the plasma membrane of the neuron. However, thesechannels are blocked by Mg2+ ions. Activation of the AMPA receptorresults in the expulsion of Mg2+ ions from the ion channels into thesynapse. When the ion channels open, and the Mg2+ ions evacuate the ionchannels, Na+ and Ca2+ ions flow into the neuron, and K+ ions flow outof the neuron.

Excitotoxicity occurs when the NMDA receptor and AMPA receptors areover-activated by the binding of excessive amounts of ligands, forexample, abnormal amounts of glutamate. The over-activation of thesereceptors causes excessive opening of the ion channels under theircontrol. This allows abnormally high levels of Ca2+ and Na+ to enter theneuron. The influx of these levels of Ca2+ and Na+ into the neuroncauses the neuron to fire more often, resulting in a rapid buildup offree radicals and inflammatory compounds within the cell. The freeradicals eventually damage the mitochondria, depleting the cell's energystores. Furthermore, excess levels of Ca2+ and Na+ ions activate excesslevels of enzymes including, but not limited to, phospholipases,endonucleases, and proteases. The over-activation of these enzymesresults in damage to the cytoskeleton, plasma membrane, mitochondria,and DNA of the sensory neuron. In some embodiments, an auris sensorycell modulating agent is a glutamate receptor antagonist that reduces orinhibits excessive neuronal firing and/or neuronal cell death. Disclosedherein, in certain embodiments, is a pharmaceutical composition for usein the treatment of a disease of the ear characterized by thedysfunction of an NMDA receptor.

Tinnitus

As used herein, “tinnitus” refers to a disorder characterized by theperception of sound in the absence of any external stimuli. In certaininstances, tinnitus occurs in one or both ears, continuously orsporadically, and is most often described as a ringing sound. It is mostoften used as a diagnostic symptom for other diseases. There are twotypes of tinnitus: objective and subjective. The former is a soundcreated in the body which is audible to anyone. The latter is audibleonly to the affected individual. Studies estimate that over 50 millionAmericans experience some form of tinnitus. Of those 50 million, about12 million experience severe tinnitus.

There are several treatments for tinnitus. Lidocaine, administered byIV, reduces or eliminates the noise associated with tinnitus in about60-80% of sufferers. Selective neurotransmitter reuptake inhibitors,such as nortriptyline, sertraline, and paroxetine, have alsodemonstrated efficacy against tinnitus. Benzodiazepines are alsoprescribed to treat tinnitus. In some embodiments, an auris sensory cellmodulating agent reduces or inhibits auris sensory cell damage and/ordeath associated with tinnitus.

Sensorineural Hearing Loss

Sensorineural hearing loss is a type of hearing loss which results fromdefects (congenital and acquired) in the vestibulocochlear nerve (alsoknown as cranial nerve VIII), or sensory cells of the inner ear. Themajority of defects of the inner ear are defects of otic hair cells.

Aplasia of the cochlea, chromosomal defects, and congenitalcholesteatoma are examples of congenital defects which can result insensorineural hearing loss. By way of non-limiting example, inflammatorydiseases (e.g. suppurative labyrinthitis, meningitis, mumps, measles,viral syphilis, and autoimmune disorders), Meniere's Disease, exposureto ototoxic drugs (e.g. aminoglycosides, loop diuretics,antimetabolites, salicylates, and cisplatin), physical trauma,presbyacusis, and acoustic trauma (prolonged exposure to sound in excessof 90 dB) can all result in acquired sensorineural hearing loss.

If the defect resulting in sensorineural hearing loss is a defect in theauditory pathways, the sensorineural hearing loss is called centralhearing loss. If the defect resulting in sensorineural hearing loss is adefect in the auditory pathways, the sensorineural hearing loss iscalled cortical deafness. In some embodiments, an auris sensory cellmodulating agent is a trophic agent (e.g., BDNF, GDNF) that promotesgrowth of auris sensory cells and reduces or reverses sensorineuralhearing loss.

Noise Induced Hearing Loss

Noise induced hearing loss (NIHL) is caused upon exposure to sounds thatare too loud or loud sounds that last an extended period of time. Longor repeated or impulse exposure to sounds at or above 85 decibels cancause hearing loss. Hearing loss may also occur from prolonged exposureto loud noises, such as loud music, heavy equipment or machinery,airplanes, gunfire or other human-based noises. NIHL causes damage tothe hair cells and/or the auditory nerve. The hair cells are smallsensory cells that convert sound energy into electrical signals thattravel to the brain. Impulse sound can result in immediate hearing lossthat may be permanent. This kind of hearing loss may be accompanied bytinnitus—a ringing, buzzing, or roaring in the ears or head—which maysubside over time. Hearing loss and tinnitus may be experienced in oneor both ears, and tinnitus may continue constantly or occasionallythroughout a lifetime. Continuous exposure to loud noise also damagesthe structure of hair cells, resulting in permanent hearing loss andtinnitus, although the process occurs more gradually than for impulsenoise.

In some embodiments, an otoprotectant can reverse, reduce or ameliorateNIHL. Examples of otoprotectants that treat or prevent NIHL include, butare not limited to, otoprotectants described herein.

Ototoxicity

Ototoxicity refers to hearing loss caused by a toxin. The hearing lossmay be due to trauma to otic hair cells, the cochlea, and/or the cranialnerve VII. Multiple drugs are known to be ototoxic. Often ototoxicity isdose-dependent. It may be permanent or reversible upon withdrawal of thedrug.

Known ototoxic drugs include, but are not limited to, the aminoglycosideclass of antibiotics (e.g. gentamicin, and amikacin), some members ofthe macrolide class of antibiotics (e.g. erythromycin), some members ofthe glycopeptide class of antibiotics (e.g. vancomycin), salicylic acid,nicotine, some chemotherapeutic agents (e.g. actinomycin, bleomycin,cisplatin, carboplatin and vincristine), and some members of the loopdiuretic family of drugs (e.g. furosemide), 6-hydroxy dopamine (6-OHDPAT), 6,7-dinitroquinoxaline-2,3-dione (DNQX) or the like.

Cisplatin and the aminoglycoside class of antibiotics induce theproduction of reactive oxygen species (“ROS”). ROS can damage cellsdirectly by damaging DNA, polypeptides, and/or lipids. Antioxidantsprevent damage of ROS by preventing their formation or scavenging freeradicals before they can damage the cell. Both cisplatin and theaminoglycoside class of antibiotics are also thought to damage the earby binding melanin in the stria vascularis of the inner ear.

Salicylic acid is classified as ototoxic as it inhibits the function ofthe polypeptide prestin. Prestin mediates outer otic hair cell motilityby controlling the exchange of chloride and carbonate across the plasmamembrane of outer otic hair cells. It is only found in the outer otichair cells, not the inner otic hair cells. Accordingly, disclosed hereinis the use of controlled release auris-compositions comprisingotoprotectants (e.g. antioxidants) to prevent, ameliorate or lessenototoxic effects of chemotherapy, including but not limited to cisplatintreatment, aminoglycoside or salicylic acid administration, or otherototoxic agents.

Endolymphatic Hydrops

Endolymphatic hydrops refers to an increase in the hydraulic pressurewithin the endolymphatic system of the inner ear. The endolymph andperilymph are separated by thin membranes which contain multiple nerves.Fluctuation in pressure stresses the membranes and the nerves theyhouse. If the pressure is great enough, disruptions may form in thesemembranes. This results in a mixing of the fluids which can lead to adepolarization blockade and transient loss of function. Changes in therate of vestibular nerve firing often leads to vertigo. Further, theorgan of Corti may also be affected. Distortions of the basilar membraneand the inner and outer hair cells can lead to hearing loss and/ortinnitus.

Causes include metabolic disturbances, hormonal imbalances, autoimmunedisease, and viral, bacterial, or fungal infections. Symptoms includehearing loss, vertigo, tinnitus, and aural fullness. Nystagmus may alsobe present. Treatment includes systemic administration ofbenzodiazepine, diuretics (to decrease the fluid pressure),corticosteroids, and/or anti-bacterial, anti-viral, or anti-fungalagents.

Labyrinthitis

Labyrinthitis is an inflammation of the labyrinths of the ear whichcontain the vestibular system of the inner ear. Causes includebacterial, viral, and fungal infections. It may also be caused by a headinjury or allergies. Symptoms of labyrinthitis include difficultymaintaining balance, dizziness, vertigo, tinnitus, and hearing loss.Recovery may take one to six weeks; however, chronic symptoms may bepresent for years.

There are several treatments for labyrinthitis. Prochlorperazine isoften prescribed as an antiemetic. Serotonin-reuptake inhibitors havebeen shown to stimulate new neural growth within the inner ear.Additionally, treatment with antibiotics is prescribed if the cause is abacterial infection, and treatment with corticosteroids and antiviralsis recommended if the condition is caused by a viral infection.

Meniere's Disease

Meniere's Disease is an idiopathic condition characterized by suddenattacks of vertigo, nausea and vomiting that may last for 3 to 24 hours,and may subside gradually. Progressive hearing loss, tinnitus and asensation of pressure in the ears accompanies the disease through time.The cause of Meniere's disease is likely related to an imbalance ofinner ear fluid homeostasis, including an increase in production or adecrease in reabsorption of inner ear fluid.

Studies of the vasopressin (VP)-mediated aquaporin 2 (AQP2) system inthe inner ear suggest a role for VP in inducing endolymph production,thereby increasing pressure in the vestibular and cochlear structures.VP levels were found to be upregulated in endolymphatic hydrops(Meniere's Disease) cases, and chronic administration of VP in guineapigs was found to induce endolymphatic hydrops. Treatment with VPantagonists, including infusion of OPC-31260 (a competitive antagonistof V2-R) into the scala tympani resulted in a marked reduction ofMeniere's disease symptoms. Other VP antagonists include WAY-140288,CL-385004, tolvaptan, conivaptan, SR 121463A and VPA 985. (Sanghi et al.Eur. Heart J. (2005) 26:538-543; Palm et al. Nephrol. Dial Transplant(1999) 14:2559-2562).

Other studies suggest a role for estrogen-related receptor β/NR3B2(ERR/Nr3b2) in regulating endolymph production, and therefore pressurein the vestibular/cochlear apparatus. Knock-out studies in micedemonstrate the role of the polypeptide product of the Nr3b2 gene inregulating endolymph fluid production. Nr3b2 expression has beenlocalized in the endolymph-secreting strial marginal cells andvestibular dark cells of the cochlea and vestibular apparatus,respectively. Moreover, conditional knockout of the Nr3b2 gene resultsin deafness and diminished endolymphatic fluid volume. Treatment withantagonists to ERR/Nr3b2 may assist in reducing endolymphatic volume,and thus alter pressure in the auris interna structures.

Other treatments may be aimed at dealing with the immediate symptoms andprevention of recurrence. Low-sodium diets, avoidance of caffeine,alcohol, and tobacco have been advocated. Medications that maytemporarily relieve vertigo attacks include antihistamines (includingmeclizine and other antihistamines), and central nervous system agents,including barbiturates and/or benzodiazepines, including lorazepam ordiazepam. Other examples of drugs that may be useful in relievingsymptoms include muscarinic antagonists, including scopolamine. Nauseaand vomiting may be relieved by suppositories containing antipsychoticagents, including the phenothiazine agent prochlorperazine.

Surgical procedures that have been used to relieve symptoms include thedestruction of vestibular and/or cochlear function to relieve vertigosymptoms. These procedures aim to either reduce fluid pressure in theinner ear and/or to destroy inner ear balance function. An endolymphaticshunt procedure, which relieves fluid pressure, may be placed in theinner ear to relieve symptoms of vestibular dysfunction. Othertreatments include gentamicin application, which when injected into theeardrum destroys sensory hair cell function, thereby eradicating innerear balance function. Severing of the vestibular nerve may also beemployed, which while preserving hearing, may control vertigo. In someembodiments, an auris sensory cell modulator promotes growth of haircells and allows a subject to regain inner ear balance function.

Meniere's Syndrome

Meniere's Syndrome, which displays similar symptoms as Meniere'sdisease, is attributed as a secondary affliction to another diseaseprocess, e.g. thyroid disease or inner ear inflammation due to syphilisinfection. Meniere's syndrome, thus, are secondary effects to variousprocess that interfere with normal production or resorption ofendolymph, including endocrine abnormalities, electrolyte imbalance,autoimmune dysfunction, medications, infections (e.g. parasiticinfections) or hyperlipidemia. Treatment of patients afflicted withMeniere's Syndrome is similar to Meniere's Disease.

Ramsay Hunt's Syndrome (Herpes Zoster Infection)

Ramsay Hunt's Syndrome is caused by a herpes zoster infection of theauditory nerve. The infection may cause severe ear pain, hearing loss,vertigo, as well as blisters on the outer ear, in the ear canal, as wellas on the skin of the face or neck supplied by the nerves. Facialmuscles may also become paralyzed if the facial nerves are compressed bythe swelling. Hearing loss may be temporary or permanent, with vertigosymptoms usually lasting from several days to weeks.

Treatment of Ramsay Hunt's syndrome includes administration of antiviralagents, including acyclovir. Other antiviral agents include famciclovirand valacyclovir. Combination of antiviral and corticosteroid therapymay also be employed to ameliorate herpes zoster infection. Analgesicsor narcotics may also be administered to relieve the pain, and diazepamor other central nervous system agents to suppress vertigo. Capsaicin,lidocaine patches and nerve blocks are optionally used. Surgery may alsobe performed on compressed facial nerves to relieve facial paralysis.

Microvascular Compression Syndrome

Microvascular compression syndrome (MCS), also called “vascularcompression” or “neurovascular compression”, is a disorder characterizedby vertigo and tinnitus. It is caused by the irritation of Cranial NerveVII by a blood vessel. Other symptoms found in subjects with MCSinclude, but are not limited to, severe motion intolerance, andneuralgic like “quick spins”. MCS is treated with carbamazepine,TRILEPTAL®, and baclofen. It can also be surgically treated.

Vestibular Neuronitis

Vestibular neuronitis, or vestibular neuropathy, is an acute, sustaineddysfunction of the peripheral vestibular system. It is theorized thatvestibular neuronitis is caused by a disruption of afferent neuronalinput from one or both of the vestibular apparatuses. Sources of thisdisruption include viral infection and acute localized ischemia of thevestibular nerve and/or labyrinth.

The most significant finding when diagnosing vestibular neuronitis isspontaneous, unidirectional, horizontal nystagmus. It is oftenaccompanied by nausea, vomiting, and vertigo. It is, however, generallynot accompanied by hearing loss or other auditory symptoms.

There are several treatments for vestibular neuronitis. H1-receptorantagonists, such as dimenhydrinate, diphenhydramine, meclizine, andpromethazine, diminish vestibular stimulation and depress labyrinthinefunction through anticholinergic effects. Benzodiazepines, such asdiazepam and lorazepam, are also used to inhibit vestibular responsesdue to their effects on the GABAA receptor. Anticholinergics, forexample scopolamine, are also prescribed. They function by suppressingconduction in the vestibular cerebellar pathways. Finally,corticosteroids (i.e. prednisone) are prescribed to ameliorate theinflammation of the vestibular nerve and associated apparatus.

Pharmaceutical Agents

Provided herein are auris sensory cell modulating agent compositions orformulations that modulate the degeneration of auris sensory cells(e.g., neurons and/or hair cells of the auris). In some embodiments,auris sensory cell modulating agent compositions or formulationsdescribed herein reduce or delay or reverse the degeneration of aurissensory cells (e.g., neurons and/or hair cells of the auris). Alsodisclosed herein are controlled release compositions for treating orameliorating hearing loss or reduction resulting from destroyed,stunted, malfunctioning, damaged, fragile or missing hairs in the innerear. Additionally provided herein are auris sensory cell modulatingagent compositions or formulations that promote the growth and/orregeneration of auris sensory cells (e.g., neurons and/or hair cells ofthe auris). In some embodiments, auris sensory cell modulating agentcompositions or formulations destroy auris sensory cells (e.g., neuronsand/or hair cells of the auris). In some embodiments, auris sensory cellmodulating agents are otoprotectants and reduce, reverse or delay damageto auris sensory cells (e.g., neurons and/or hair cells of the auris).

Otic and vestibular disorders have causes and symptoms that areresponsive to the pharmaceutical agents disclosed herein, or otherpharmaceutical agents. Auris sensory cell modulating agents which arenot disclosed herein but which ameliorate or eradicate otic disordersare expressly included and intended within the scope of the embodimentspresented.

Moreover, pharmaceutical agents which have been previously shown to betoxic, harmful or non-effective during systemic or localized applicationin other organ systems, for example through toxic metabolites formedafter hepatic processing, toxicity of the drug in particular organs,tissues or systems, through high levels needed to achieve efficacy,through the inability to be released through systemic pathways orthrough poor pK characteristics, are useful in some embodiments herein.Accordingly, pharmaceutical agents which have limited or no systemicrelease, systemic toxicity, poor pK characteristics or combinationsthereof are contemplated within the scope of the embodiments disclosedherein.

The auris sensory cell modulating agent formulations disclosed hereinare optionally targeted directly to otic structures where treatment isneeded; for example, one embodiment contemplated is the directapplication of the auris sensory cell modulating agent formulationsdisclosed herein onto the round window membrane or the crista fenestraecochlea of the auris interna, allowing direct access and treatment ofthe auris interna, or inner ear components. In other embodiments, theauris sensory cell modulating agent formulation disclosed herein isapplied directly to the oval window. In yet other embodiments, directaccess is obtained through microinjection directly into the aurisinterna, for example, through cochlear microperfusion. Such embodimentsalso optionally comprise a drug delivery device, wherein the drugdelivery device delivers the auris sensory cell modulating agentformulations through use of a needle and syringe, a pump, amicroinjection device, an auris-acceptable in situ forming spongymaterial or any combination thereof.

Some pharmaceutical agents, either alone or in combination, areototoxic. For example, some chemotherapeutic agents, includingactinomycin, bleomycin, cisplatin, carboplatin and vincristine; andantibiotics, including erythromycin, gentamicin, streptomycin,dihydrostreptomycin, tobramycin, netilmicin, amikacin, neomycin,kanamycin, etiomycin, vancomycin, metronidizole, capreomycin, are mildlyto very toxic, and affect the vestibular and cochlear structuresdifferentially. However, in some instances, the combination of anototoxic drug, for example cisplatin, gentamicin, in combination with anotoprotectant is lessens the ototoxic effects of the drug. Moreover, thelocalized application of the potentially ototoxic drug also lessens thetoxic effects that would otherwise occur through systemic applicationthrough the use of lower amounts with maintained efficacy, or the use oftargeted amounts for a shorter period of time.

In some embodiments, the auris sensory cell modulating agentformulations disclosed herein further comprise otoprotectants thatreduce, inhibit or ameliorate the ototoxicity of agents such aschemotherapeutic agents and/or antibiotics as described herein, orreduce, inhibit or ameliorate the effects of other environmentalfactors, including excessive noise and the like. Examples ofotoprotectants include, and are not limited to, otoprotectants describedherein, thiols and/or thiol derivatives and/or pharmaceuticallyacceptable salts, or derivatives (e.g. prodrugs) thereof.

Otoprotectants allow for the administration of ototoxic agents and/orantibiotics at doses that are higher than maximal toxic doses; theototoxic agents and/or antibiotics would otherwise be administered atlower doses due to ototoxicity. An otoprotectant, when optionallyadministered by itself, also allows for the amelioration, reduction orelimination of the effect of environmental factors that contribute toloss of hearing and attendant effects, including but not limited tonoise-induced hearing loss and tinnitus.

The amount of otoprotectant in any formulation described herein on amole:mole basis in relation to the ototoxic chemotherapeutic agent (e.g.cis platin) and/or an ototoxic antibiotic (e.g. gentamicin) is in therange of from about 5:1 to about 200:1, from about 5:1 to about 100:1,or from about 5:1 to about 20:1. The amount of otoprotectant in anyformulation described herein on a molar basis in relation to theototoxic chemotherapeutic agent (e.g. cis platin) and/or an ototoxicantibiotic (e.g. gentamicin) is about 50:1, about 20:1 or about 10:1.Any auris sensory cell modulating agent formulation described hereincomprises from about 10 mg/ml to about 50 mg/ml, from about 20 mg/ml toabout 30 mg/ml, or from 10 mg/mL to about 25 mg/ml of otoprotectant.

Moreover, some pharmaceutical excipients, diluents or carriers arepotentially ototoxic. For example, benzalkonium chloride, a commonpreservative, is ototoxic and therefore potentially harmful ifintroduced into the vestibular or cochlear structures. In formulating acontrolled release auris sensory cell modulating agent formulation, itis advised to avoid or combine the appropriate excipients, diluents orcarriers to lessen or eliminate potential ototoxic components from theformulation, or to decrease the amount of such excipients, diluents orcarriers. Optionally, a controlled release auris sensory cell modulatingagent formulation includes otoprotective agents, such as antioxidants,alpha lipoic acid, calcium, fosfomycin or iron chelators, to counteractpotential ototoxic effects that may arise from the use of specifictherapeutic agents or excipients, diluents or carriers.

Amifostine

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents which rescue neurons and otic hair cellsfrom cisplatin-induced ototoxicity.

Amifostine (also known as WR-2721, or ETHYOL®) is an otoprotectiveagent. In certain instances, it prevents or ameliorates the damage toneuron and otic hair cells caused by cisplatin. In certain instances,doses at or above 40 mg/kg are needed to protect against or amelioratethe ototoxic effects of cisplatin.

Salicylic Acid

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of salicylic acid. In certain instances, salicyclicacid is an antioxidant and when administered before treatment with anaminoglycoside, it protects otic hair cells and spiral ganglion neuronsfrom aminoglycoside ototoxicity.

Modulation of Atoh/Math1

Contemplated for use with the formulations disclosed herein are agentsthat promote the growth and/or regeneration of neurons and/or otic haircells. Atoh1 is a transcription factor which binds to an E-box. Incertain instances, it is expressed during the development of the haircells of the vestibular and auditory systems. In certain instances, micewith Atoh1 knocked-out did not develop otic hair cells. In certaininstances, adenoviruses expressing Atoh1 stimulate the growth and/orregeneration of otic hair cells in guinea pigs treated with ototoxicantibiotics. Accordingly, some embodiments incorporate modulation of theAtoh1 gene.

In some embodiments, a subject is administered a vector engineered tocarry the human Atoh1 gene (the “Atoh1 vector”). For disclosures oftechniques for creating the Atoh1 vector see U.S. Pub. No.2004/02475750, which is hereby incorporated by reference for thosedisclosures. In some embodiments, the Atoh1 vector is a retrovirus. Insome embodiments, the Atoh1 vector is not a retrovirus (e.g. it is anadenovirus; a lentivirus; or a polymeric delivery system such asMETAFECTENE, SUPERFECT®, EFFECTENE®, or MIRUS TRANSIT).

In some embodiments, the Atoh1 vector is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the inner ear. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the cochlea, the organ of Corti, the vestibular labyrinth, or acombination thereof.

In certain instances, after administration of the Atoh1 vector, theAtoh1 vector infects the cells at the site of administration (e.g. thecells of cochlea, organ of Corti, and/or the vestibular labyrinth). Incertain instances the Atoh1 sequence is incorporated into the subject'sgenome (e.g. when the Atoh1 vector is a retrovirus). In certaininstances the therapy will need to be periodically re-administered (e.g.when the Atoh1 vector is not a retrovirus). In some embodiments, thetherapy is re-administered annually. In some embodiments, the therapy isre-administered semi-annually. In some embodiments, the therapy isre-administered when the subject's hearing loss is moderate (i.e. thesubject cannot consistently hear frequencies less than 41 db to 55 dB)to profound (i.e. the subject cannot consistently hear frequencies lessthan 90 dB).

In some embodiments, a subject is administered the Atoh1 polypeptide. Insome embodiments, the Atoh1 polypeptide is incorporated intocontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel. In some embodiments, the auris-acceptable microsphere ormicroparticle, hydrogel, liposome, or thermoreversible gel. In someembodiments, the auris-acceptable microsphere, hydrogel, liposome,paint, foam, in situ forming spongy material, nanocapsule or nanosphereor thermoreversible gel is injected into the inner ear. In someembodiments, the auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the cochlea, the organ of Corti, the vestibularlabyrinth, or a combination thereof. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is placed incontact with the round window membrane.

In some embodiments, a subject is administered a pharmaceuticallyacceptable agent which modulates the expression of the Atoh1 gene oractivity of the Atoh1 polypeptide. In some embodiments, the expressionof the Atoh1 gene or activity of the Atoh1 polypeptide is up-regulated.In some embodiments, the expression of the Atoh1 gene or activity of theAtoh1 polypeptide is down-regulated.

In certain instances, a compound which agonizes or antagonizes Atoh1 isidentified (e.g. by use of a high throughput screen). In someembodiments, a construct is designed such that a reporter gene is placeddownstream of an E-box sequence. In some embodiments, the reporter geneis luciferase, CAT, GFP, β-lactamase or β-galactosidase. In certaininstances, the Atoh1 polypeptide binds to the E-box sequence andinitiates transcription and expression of the reporter gene. In certaininstances, an agonist of Atoh1 aids or facilitates the binding of Atoh1to the E-box sequence, thus increasing transcription and expression ofthe reporter gene relative to a pre-determined baseline expressionlevel. In certain instances, an antagonist of Atoh1 blocks the bindingof Atoh1 to the E-box, thus decreasing transcription and expression ofthe reporter gene relative to a pre-determined baseline expressionlevel.

BRN-3 Modulators

Contemplated for use with the formulations disclosed herein are agentsthat promote the growth and/or regeneration of neurons and/or otic haircells. BRN-3 is a group of transcription factors that include, but arenot limited to, BRN-3a, BRN-3b, and BRN-3c. In certain instances, theyare expressed in postmitotic hair cells. In certain instances, the haircells of mice with BRN-3c knocked-out did not develop stereocilia and/orunderwent cell death. In certain instances, BRN3 genes regulate thedifferentiation of inner ear supporting cells into inner ear sensorycells. Accordingly, some embodiments incorporate modulation of the BRN3genes, and/or polypeptides.

In some embodiments, a subject is administered a vector engineered tocarry a human BRN-3 gene (the “BRN3 vector”). In some embodiments, theBRN3 vector is a retrovirus. In some embodiments, the BRN3 vector is nota retrovirus (e.g. it is an adenovirus; a lentivirus; or a polymericdelivery system such as METAFECTENE, SUPERFECT®, EFFECTENE®, or MIRUSTRANSIT).

In some embodiments, the subject is administered the BRN3 vector before,during, or after exposure to an ototoxic agent (e.g. an aminoglycosideor cisplatin), or a sound of sufficient loudness to induce acoustictrauma.

In some embodiments, the BRN3 vector is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the inner ear. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the cochlea, the organ of Corti, the vestibular labyrinth, or acombination thereof.

In certain instances, after administration of the BRN3 vector, the BRN3vector infects the cells at the site of administration (e.g. the cellsof cochlea, organ of Corti, and/or the vestibular labyrinth). In certaininstances the BRN3 sequence is incorporated into the subject's genome(e.g. when the BRN3 vector is a retrovirus). In certain instances thetherapy will need to be periodically re-administered (e.g. when the BRN3vector is not a retrovirus).

In some embodiments, a subject is administered a BRN3 polypeptide. Insome embodiments, the BRN3 polypeptide is incorporated intocontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel. In some embodiments, the auris-acceptable microsphere ormicroparticle, hydrogel, liposome, or thermoreversible gel. In someembodiments, the auris-acceptable microsphere, hydrogel, liposome,paint, foam, in situ forming spongy material, nanocapsule or nanosphereor thermoreversible gel is injected into the inner ear. In someembodiments, the auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the cochlea, the organ of Corti, the vestibularlabyrinth, or a combination thereof. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is placed incontact with the round window membrane.

In some embodiments, a subject is administered a pharmaceuticallyacceptable agent which modulates the expression of the BRN3 gene oractivity of the BRN3 polypeptide. In some embodiments, the expression ofthe BRN3 gene or activity of the BRN3 polypeptide is up-regulated. Insome embodiments, the expression of the BRN3 gene or activity of theBRN3 polypeptide is down-regulated.

In some embodiments, a compound which agonizes or antagonizes BRN3 isidentified (e.g. by use of a high throughput screen). In someembodiments, a construct is designed such that a reporter gene is placeddownstream of a BRN3 binding site. In some embodiments, the BRN3 bindingsite has the sequence ATGAATTAAT (SBNR3). In some embodiments, thereporter gene is luciferase, CAT, GFP, β-lactamase or β-galactosidase.In certain instances, the BRN3 polypeptide binds to the SBNR3 sequenceand initiates transcription and expression of the reporter gene. Incertain instances, an agonist of BRN3 aids or facilitates the binding ofBRN3 to the SBNR3 sequence, thus increasing transcription and expressionof the reporter gene relative to a pre-determined baseline expressionlevel. In certain instances, an antagonist of BRN3 blocks the binding ofBRN3 to the SBNR3, thus decreasing transcription and expression of thereporter gene relative to a pre-determined baseline expression level.

Carbamates

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. In certain instances, carbamatecompounds protect neurons and otic hair cells from glutamate-inducedexcitotoxicity. Accordingly, some embodiments incorporate the use ofcarbamate compounds. In some embodiments, the carbamate compounds are2-phenyl-1,2-ethanediol monocarbomates and dicarbamates, derivativesthereof, and/or combinations thereof.

Gamma-Secretase Inhibitors

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents which inhibit Notch1 signaling. Notch1 isa transmembrane polypeptide which participates in cell development. Insome embodiments, the agents which inhibit Notch1 signaling areγ-secretase inhibitors. In certain instances, the inhibition of Notch1by a γ-secretase inhibitor, following treatment with an ototoxic agent,results in the production/growth of otic hair cells. In someembodiments, the γ-secretase inhibitor is LY450139 (hydroxylvalerylmonobenzocaprolactam), L685458(1S-benzyl-4R[1-[1-S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl}carbamicacid tert-butyl ester); LY411575(N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[bid]azepin-7yl]-L-alaninamide),MK-0752 (Merck), tarenflurbil, and/or BMS-299897(2-[(1R)-1-[[(4-chlorophenyl)sulfony](2,5-difluorophenyl)amino]ethyl]-5-fluorobenzenepropanoicacid).

Glutamate-Receptor Modulators

Provided herein are methods of treating an otic disorder characterizedby the dysregulation (e.g., over-activation or over-stimulation) of aglutamate receptor. In some embodiments, a method disclosed hereincomprises administering to an individual in need thereof a compositioncomprising a glutamate receptor antagonist. Glutamate receptorantagonists which are not disclosed herein but which are useful for theamelioration or eradication of otic disorders are expressly included andintended within the scope of the embodiments presented.

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents which modulate glutamate receptors. Insome embodiments, the glutamate receptor is the AMPA receptor, and/or agroup II or III mGlu receptor. In some embodiments, the glutamatereceptor is the NMDA receptor. In some embodiments, the glutamatereceptor modulator is a glutamate receptor antagonist. In someembodiments, the glutamate receptor antagonist is a non-competitiveantagonist. In some embodiments, the glutamate receptor antagonist is asmall molecule.

In some embodiments, the agent that modulates the AMPA receptor is anAMPA receptor antagonist. In some embodiments, the agent whichantagonizes the AMPA receptors is CNQX(6-cyano-7-nitroquinoxaline-2,3-dione); NBQX(2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione); DNQX(6,7-dinitroquinoxaline-2,3-dione); kynurenic acid;2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-[f]quinoxaline; or combinationsthereof.

In some embodiments, the glutamate receptor antagonist is an NMDAreceptor antagonist. In some embodiments, the agent that modulates theNMDA receptor is an NMDA receptor antagonist. In some embodiments, theglutamate receptor antagonist 1-aminoadamantane; dextromethorphan;dextrorphan; ibogaine; ifenprodil; (S)-ketamine; (R)-ketamine;memantine; dizocilpine (MK-801); gacyclidine; AM-101; traxoprodil;D-2-amino-5-phosphonopentanoic acid (D-AP5);3-((±)2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP);conantokin; 7-chlorokynurenate (7-CK); licostinel; nitrous oxide;phencyclidine; riluzole; tiletamine; aptiganel; remacimide; DCKA (5;7-dichlorokynurenic acid); kynurenic acid; 1-aminocyclopropanecarboxylicacid (ACPC); AP7 (2-amino-7-phosphonoheptanoic acid); APV(R-2-amino-5-phosphonopentanoate); CPPene(3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid);(+)-(1S;2S)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-pro-panol;(1S,2S)-1-(4-hydroxy-3-methoxyphenyl)-2-(4-hydroxy-4-phenylpiperi-dino)-1-propanol;(3R,4S)-3-(4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl-)-chroman-4,7-diol;(1R*;2R*)-1-(4-hydroxy-3-methylphenyl)-2-(4-(4-fluoro-phenyl)-4-hydroxypiperidin-1-yl)-propan-1-ol-mesylate;or combinations thereof. In some embodiments, the NMDA receptorantagonist is an arylcycloalkylamine. In some embodiments, the NMDAreceptor antagonist is (S)-ketamine or a salt thereof. In someembodiments, the NMDA receptor antagonist is a chinazoline. In someembodiments, the NMDA receptor antagonist is 7-CK or a salt thereof. Insome embodiments, the NMDA receptor antagonist is AM-101 or a saltthereof.

In some embodiments, the glutamate receptor antagonist is a peptide. Insome embodiments, the glutamate receptor antagonist is a fusion peptidecomprising (a) a transporter peptide, and (b) a peptide inhibitinginteraction of an NMDA receptor with NMDA receptor interacting proteins.As used herein, a “transporter peptide” means a peptide that promotespeptide penetration into cells and tissues. In some embodiments, atransporter peptide is TAT.

In some embodiments, the glutamate receptor antagonist is a fusionpeptide comprising (a) a TAT peptide, and (b) a peptide inhibitinginteraction of an NMDA receptor with NMDA receptor interacting proteins.In some embodiments, the glutamate receptor antagonist is a fusionpeptide comprising (a) a (D)-TAT peptide, and (b) a peptide inhibitinginteraction of an NMDA receptor with NMDA receptor interacting proteins.In some embodiments, the glutamate receptor antagonist is a fusionpeptide comprising (a) a transporter peptide, and (b) an NR2B9c peptide.In some embodiments, the glutamate receptor antagonist is a fusionpeptide comprising (a) a transporter peptide, and (b) a (D) —NR2B9cpeptide. In some embodiments, the glutamate receptor antagonist is afusion peptide comprising (a) a (D)-TAT peptide, and (b) a (D)-NR2B9cpeptide. In some embodiments, the glutamate receptor antagonist is afusion peptide comprising (a) a transporter peptide, and (b) a(L)-NR2B9c peptide.

In certain instances, the over-activation of the AMPA and NMDA glutamatereceptors by the binding of excessive amounts of glutamate, results inthe excessive opening of the ion channels under their control. Incertain instances, this results in abnormally high levels of Ca2+ andNa+ entering the neuron. In certain instances, the influx of Ca2+ andNa+ into the neuron activates multiple enzymes including, but notlimited to, phospholipases, endonucleases, and proteases. In certaininstances, the over-activation of these enzymes results in damage to thecytoskeleton, plasma membrane, mitochondria, and DNA of the neuron.Further, in certain instances, the transcription of multiplepro-apoptotic genes and anti-apoptotic genes are controlled by Ca2+levels.

The mGlu receptors, unlike the AMPA and NMDA receptors, do not directlycontrol an ion channel. However, in certain instances, they indirectlycontrol the opening of ion channels by the activation of biochemicalcascades. The mGlu receptors are divided into three groups. In certaininstances, the members of groups II and III reduce or inhibitpost-synaptic potentials by preventing or decreasing the formation ofcAMP. In certain instances, this causes a reduction in the release ofneurotransmitters, especially glutamate. GRM7 is the gene which encodesthe mGlu7 receptor, a group III receptor. In certain instances, theagonism of mGlu7 results in a decrease in synaptic concentrations ofglutamate. This ameliorates glutamate excitotoxicity.

In some embodiments, the glutamate receptor is a group II mGlu receptor.In some embodiments, the agent which modulates the group II mGlureceptor is a group II mGlu receptor agonist. In some embodiments, thegroup II mGlu receptor agonist is LY389795((−)-2-thia-4-aminobicyclo-hexane-4,6-dicarboxylate); LY379268((−)-2-oxa-4-aminobicyclo-hexane-4,6-dicarboxylate); LY354740((+)-2-aminobicyclo-hexane-2,6dicarboxylate); DCG-IV((2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine); 2R,4R-APDC(2R,4R-4-aminopyrrolidine-2,4-dicarboxylate), (S)-3C4HPG((S)-3-carboxy-4-hydroxyphenylglycine); (S)-4C3HPG((S)-4-carboxy-3-hydroxyphenylglycine); L-CCG-I((2S,1′S,2′S)-2-(carboxycyclopropyl)glycine); and/or combinationsthereof.

In some embodiments, the mGlu receptor is a group III mGlu receptor. Insome embodiments, the group III mglu receptor is mGlu7. In someembodiments, the agent which modulates the group III mglu receptor is agroup III mglu receptor agonist. In some embodiments, the group III mGlureceptor agonist is ACPT-I((1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid); L-AP4(L-(+)-2-Amino-4-phosphonobutyric acid); (S)-3,4-DCPG((S)-3,4-dicarboxyphenylglycine); (RS)-3,4-DCPG((RS)-3,4-dicarboxyphenylglycine); (RS)-4-phosphonophenylglycine((RS)PPG); AMN082 (,N′-bis(diphenylmethyl)-1,2-ethanediaminedihydrochloride); DCG-IV((2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine); and/orcombinations thereof. In some embodiments, the mGlu receptor is mGlu7.In some embodiments, the agonist of mGlu7 is AMN082. In someembodiments, the mGlu receptor modulator is3,5-Dimethylpyrrole-2,4-dicarboxylic acid 2-propyl ester4-(1,2,2-trimethyl-propyl) ester (3,5-dimethyl PPP);3,3′-difluorobenzaldazine (DFB), 3,3′-dimlethoxybenzaldazine (DMeOB),3,3′-dichlorobenzaldazine (DCB) and other allosteric modulators ofmGluR5 disclosed in Mol. Pharmacol. 2003, 64, 731-740;(E)-6-methyl-2-(phenyldiazenyl)pyridin-3-ol (SIB 1757);(E)-2-methyl-6-styrylpyridine (SIB 1893);2-methyl-6-(phenylethynyl)pyridine (MPEP),2-methyl-4-((6-methylpyridin-2-yl)ethynyl)thiazole (MTEP);7-(Hydroxyimino)cyclopropa[b]chromen-1α-carboxylate ethyl ester(CPCCOEt),N-cyclohexyl-3-methylbenzo[d]thiazolo[3,2-a]imidazole-2-carboxamide(YM-298198), tricyclo[3.3.3.1]nonanyl quinoxaline-2-carboxamide (NPS2390); 6-methoxy-N-(4-methoxyphenyl)quinazolin-4-amine (LY 456239);mGluR1 antagonists disclosed in WO2004/058754 and WO2005/009987;2-(4-(2,3-dihydro-1H-inden-2-ylamino)-5,6,7,8-tetrahydroquinazolin-2-ylthio)ethanol;3-(5-(pyridin-2-yl)-2H-tetrazol-2-yl)benzonitrile,2-(2-methoxy-4-(4-(pyridin-2-yl)oxazol-2-yl)phenyl)acetonitrile;2-(4-(benzo[d]oxazol-2-yl)-2-methoxyphenyl)acetonitrile;6-(3-methoxy-4-(pyridin-2-yl)phenyl)imidazo[2,1-b]thiazole;(S)-(4-fluorophenyl)(3-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)methanone(ADX47273) and/or combinations thereof.

In some embodiments, a glutamate receptor modulator is a nootropicagent. Contemplated for use with the formulations disclosed herein arenootropic agents that modulate neuronal signalling by activatingglutamate receptors. In some instances, nootropic agents treat orameliorate hearing loss (e.g., NIHL) or tinnitus. Accordingly, someembodiments incorporate the use of nootropic agents including, and notlimited to, piracetam, Oxiracetam, Aniracetam, Pramiracetam,Phenylpiracetam (Carphedon), Etiracetam, Levetiracetam, Nefiracetam,Nicoracetam, Rolziracetam, Nebracetam, Fasoracetam, Coluracetam,Dimiracetam, Brivaracetam, Seletracetam, and/or Rolipram for thetreatment of NIHL or tinnitus.

Trophic Agents

Contemplated for use with the formulations disclosed herein are agentsthat reduce or delay the degeneration of neurons and/or hair cells ofthe auris. In some embodiments, contemplated for use with thecompositions described herein are agents that are trophic agents, e.g.,agents that promote the growth of tissue and/or neurons and/or haircells of the auris. Also contemplated for use with the compositionsdescribed herein are agents for treating or ameliorating hearing loss orreduction resulting from destroyed, stunted, malfunctioning, damaged,fragile or missing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of trophic agents which promote the survival ofneurons and otic hair cells, and/or the growth of neurons and otic haircells. In some embodiments, the trophic agent which promotes thesurvival of otic hair cells is a growth factor. In some embodiments, thegrowth factor is a neurotroph. In certain instances, neurotrophs aregrowth factors which prevent cell death, prevent cell damage, repairdamaged neurons and otic hair cells, and/or induce differentiation inprogenitor cells. In some embodiments, the neurotroph is brain-derivedneurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glialcell-line derived neurotrophic factor (GDNF), neurotrophin-3,neurotrophin-4, and/or combinations thereof. In some embodiments, thegrowth factor is a fibroblast growth factor (FGF), an insulin-likegrowth factor (IGF), an epidermal growth factor (EGF), a platlet-derivedgrowth factor (PGF) and/or agonists thereof. In some embodiments, thegrowth factor is an agonist of the fibroblast growth factor (FGF)receptor, the insulin-like growth factor (IGF) receptor, the epidermalgrowth factor (EGF) receptor, and/or the platlet-derived growth factor.In some embodiments, the growth factor is hepatocyte growth factor.

In some embodiments, the trophic agent and/or neurotroph is BDNF. Insome embodiments, the trophic agent and/or neurotroph is GDNF. Incertain instances, BDNF and GDNF are neurotrophs that promote thesurvival of existing neurons (e.g. spiral ganglion neurons), and otichair cells by repairing damaged cells, inhibiting the production of ROS,and/or inhibiting cell death. In certain instances, it also promotes thedifferentiation of neural and otic hair cell progenitors. Further, incertain instances, it protects the Cranial Nerve VII from degeneration.In some embodiments, BDNF is administered in conjunction with fibroblastgrowth factor.

In some embodiments, the neurotroph is neurotrophin-3. In certaininstances, neurotrophin-3 promotes the survival of existing neurons andotic hair cells, and promotes the differentiation of neural and otichair cell progenitors. Further, In certain instances, it protects theVII nerve from degeneration.

some embodiments, the neurotroph is CNTF. In certain instances, CNTFpromotes the synthesis of neurotransmitters and the growth of neuritis.In some embodiments, CNTF is administered in conjunction with BDNF.

In some embodiments, the trophic agent and/or neurotroph is GDNF. Incertain instances, GDNF expression is increased by treatment withototoxic agents. Further, in certain instances, cells treated withexogenous GDNF have higher survival rates after trauma than untreatedcells.

In some embodiments, the trophic agent and/or growth factor is anepidermal growth factor (EGF). In some embodiments, the EGF is heregulin(HRG). In certain instances, HRG stimulates the proliferation ofutricular sensory epithelium. In certain instances, HRG-bindingreceptors are found in the vestibular and auditory sensory epithelium.

In some embodiments, the trophic agent and/or growth factor is aninsulin-like growth factor (IGF). In some embodiments, the IGF is IGF-1.In some embodiments, the IGF-1 is mecasermin. In certain instances,IGF-1 attenuates the damage induced by exposure to an aminoglycoside. Incertain instances, IGF-1 stimulates the differentiation and/ormaturation of cochlear ganglion cells.

In some embodiments, the FGF receptor agonist is FGF-2. In someembodiments, the IGF receptor agonist is IGF-1. Both the FGF and IGFreceptors are found in the cells comprising the utricle epithelium.

In some embodiments, the growth factor is hepatocyte growth factor(HGF). In some instances, HGF protects cochlear hair cells fromnoise-induced damage and reduces noise-exposure-caused ABR thresholdshifts.

Also contemplated for use in the otic formulations described herein aregrowth factors including Erythropoietin (EPO), Granulocyte-colonystimulating factor (G-CSF), Granulocyte-macrophage colony stimulatingfactor (GM-CSF), Growth differentiation factor-9 (GDF9), Insulin-likegrowth factor (IGF), Myostatin (GDF-8), Platelet-derived growth factor(PDGF), Thrombopoietin (TPO), Transforming growth factor alpha (TGF-α),Transforming growth factor beta (TGF-β), Vascular endothelial growthfactor (VEGF) or combinations thereof. Also contemplated for use in theotic compositions described herein are trophic factors includingantioxidants and/or vitamins that are described herein.

Anti-Intercellular Adhesion Molecule-1 Antibody

Contemplated for use with the formulations disclosed herein areantibodies to anti-intercellular adhesion molecule (ICAM). In someinstances, ICAM blocks the cascade of reactive oxygen species associatedwith exposure to noise. In some instances modulation of the cascade ofreactive oxygen species associated with exposure to noise ameliorates orreduces the degeneration of neurons and/or hair cells of the auris.Accordingly, some embodiments incorporate the use of agents that areantibodies to ICAMs (e.g., anti-ICAM-1 Ab, anti-ICAM-2 Ab or the like).

Otoprotectants

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of otoprotectants. In some embodiments, anotoprotectant is a glutamate receptor antagonist as described herein. Insome embodiments, an otoprotectant is a corticosteroid as describedherein. In some embodiments, otoprotectants are agents that modulateglutathione peroxidase (GPx). The enzyme GPx reduces reactive oxygenspecies (ROS) in the cohclea and maintains the health of neurons and/orhair cells in the inner ear. Modulators of GPx include and are notlimited to glutathione peroxidase mimics such as2-phenyl-1,2-benzoisoselenazol-3 (2H)-one (ebselen, SPI-1005),6A,6B-diseleninic acid-6A′,6B′-selenium bridged β-cyclodextrin(6-diSeCD), and 2,2′-diseleno-bis-β-cyclodextrin (2-diSeCD).

In some embodiments, the use of otoprotectants reduces or ameliorateshearing loss or reduction in hearing resulting from destroyed, stunted,malfunctioning, damaged, fragile or missing hairs in the inner ear.Otoprotectants include, but are not limited to, D-methionine,L-methionine, ethionine, hydroxyl methionine, methioninol, amifostine,mesna (sodium 2-sulfanylethanesulfonate), a mixture of D and Lmethionine, N-acetyl methionine (NAM), normethionine, homomethionine,S-adenosyl-L-methionine, diethyldithiocarbamate, ebselen(2-phenyl-1,2-benzisoselenazol-3(2H)-one), sodium thiosulfate, AM-111 (acell permeable JNK inhibitor, (Laboratories Auris SAS)),N-acetyl-DL-methionine, S-adenosylmethionine, cysteine, homocysteine,cysteamine, N-acetylcysteine (NAC), glutathione, glutathione ethylester,glutathione diethylester, glutathione triethylester, cysteamine,cystathione, N,N′-diacetyl-L-cystine (DiNAC),2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)-thiazolidine-4(R)-carboxylicacid (RibCys), 2-alkylthiazolidine2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidine (RibCyst), and2-oxo-L-thiazolidine-4-carboxylic acid (OTCA), salicylic acid,leucovorin, leucovorin calcium, dexrazoxane, piracetam, Oxiracetam,Aniracetam, Pramiracetam, Phenylpiracetam (Carphedon), Etiracetam,Levetiracetam, Nefiracetam, Nicoracetam, Rolziracetam, Nebracetam,Fasoracetam, Coluracetam, Dimiracetam, Brivaracetam, Seletracetam,Rolipramand or combinations thereof.

In some embodiments, otoprotectants include xanthine oxidase inhibitors.Non-limiting examples of xanthine odixase inhibitors includeallopurinol; 1-methylallopurinol; 2-methylallopurinol;5-methylallopurinol; 7-methylallopurinol; 1,5-dimethylallopurinol;2,5-dimethylallopurinol; 1,7-dimethylallopurinol;2,7-dimethylallopurinol; 5,7-dimethylallopurinol;2,5,7-trimethylallopurinol; 1-ethoxycarbonylallopurinol; and1-ethoxycarbonyl-5-methylallopurinol.

In some embodiments, otoprotectants are used in combination withtoxicants.

Modulators of Hair Cell Regeneration

Contemplated for use with the formulations disclosed herein are agentsthat modulate the regeneration of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. In some embodiments, an auris sensorycell modulator allows for proliferation and/or regeneration of aurishair cells and/or supporting cells. Accordingly, some embodimentsincorporate the use of cyclin-dependent kinase (CDK) modulators. In someembodiments, CDK modulators are p27Kip1 modulators. p27Kip1 mediatessensory hair cell regeneration in the organ of Corti. In some instances,sensory hair cells (e.g., short hair cells) of the inner ear areregenerated by stimulation of proliferation of supporting cells (e.g.Hansen's cells, Deiter's cells and/or Pillar's cells or the like). Insome instances modulators of the cyclin-dependent kinase p27Kip1 areantisense molecules (e.g., siRNA molecules) or peptide molecules (e.g.,endogenous ligands for p27Kip1) that modulate the activity of p27Kip1.

In some embodiments, a formulation described herein comprises a nucleicacid and/or transcription factor (e.g., POU4F1, POU4F2, POU4F3, Brn3a,Brn3b and/or Brn3c or the like) capable of stimulating the formation ofinner ear sensory hair cells or inner ear support cells. Non-limitingexamples of such nucleic acid molecules and/or transcription factorsinclude and are not limited to molecules described in US Appl. Pub. Nos.20070041957 and 20030203482, which disclosure described therein isincorporated herein by reference.

Immune System Cells

Contemplated for use with the formulations disclosed herein are agentsthat reduce, reverse or dealy the degeneration of neurons and/or haircells of the auris, and agents for treating or ameliorating hearing lossor reduction resulting from destroyed, stunted, malfunctioning, damaged,fragile or missing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of cells which participate in the repair of otichair cells and neurons. In some embodiments, the cells which participatein the repair of otic hair cells and neurons are macrophages, microglia,and/or microglia-like cells. In certain instances, the concentration ofmacrophages and microglia increase in ears damaged by treatment withototoxic agents. In certain instances, microglia-like cells eliminatewaste from ototoxic antibiotic neomycin.

Ototoxic Agents and Toxicants

Contemplated for use with the formulations disclosed herein are agentsthat destroy neurons and/or otic hair cells. Accordingly, someembodiments incorporate the use of agents which fatally damage and/orinduce death in the neurons and/or otic hair cells of the auris. In someembodiments, death of auris sensory cells (e.g., hair cells) treatssymptoms (e.g., vertigo) associated with any otic disease or conditiondescribed herein. In some embodiments, toxicants induce chemical lesionsin the ear that alleviate symptoms such as, by way of example, vertigo.In some embodiments, the agents which fatally damage and/or induce deathin the neurons and/or otic hair cells of the auris are theaminoglycoside antibiotics (e.g. gentamicin, and amikacin), themacrolide antibiotics (e.g. erythromycin), the glycopeptide antibiotics(e.g. vancomycin), the loop diuretics (e.g. furosemide), nicotine,6-hydroxy dopamine (6-OH DPAT), 6,7-dinitroquinoxaline-2,3-dione (DNQX)or the like. In some embodiments, a composition described hereincomprises a toxicant that is used to treat vertigo by selectivelydestroying hair cells in the ear. In some of such embodiments, an oticcomposition comprising a toxicant is advantageous; such a compositionprovides therapeutic benefit for treatment of vertigo because it isadministered non-systemically to the ear and does not cause side effectsassociated with systemic administration of a toxicant.

In some embodiments, a composition described herein is useful inpre-clinical animal studies (e.g., animal guinea pig model studies). Insome instances, a composition described herein comprising a toxicant isused for induction of chemical lesions in the ear of an animal. In someinstances, such an animal is used for testing therapeutic efficacy ofcompositions described herein in animal models.

Retinoblastoma Protein Modulation

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and/or hair cells of theauris, promote the growth of neurons and/or hair cells of the auris, andagents for treating or ameliorating hearing loss or reduction resultingfrom destroyed, stunted, malfunctioning, damaged, fragile or missinghairs in the inner ear. Further contemplated herein are agents thatdestroy neurons and/or otic hair cells. Accordingly, some embodimentsincorporate the use of agents that modulate retinoblastoma protein(pRB). pRB is a member of the pocket protein family. It is encoded bythe RB1 gene. In certain instances, it inhibits transition from G1 to Sphase by binding to and inactivating the E2f family of transcriptionfactors. In certain instances, it also regulates differentiation, andsurvival of hair cells. In certain instances, pRB knock-out micedemonstrate increased proliferation of hair cells.

In some embodiments, the agent that modulates one or more of the pRB isan agonist of pRB. In some embodiments, the agent that modulates one ormore of the pRB is an antagonist of pRB. In certain instances, acompound which agonizes or antagonizes pRB is identified (e.g. by use ofa high throughput screen). In some embodiments, a construct is designedsuch that a reporter gene is placed downstream of an E2F bindingsequence. In some embodiments, the binding sequence is TTTCGCGC. In someembodiments, the reporter gene is luciferase, CAT, GFP, β-lactamase orβ-galactosidase. In certain instances, E2f binds to the binding sequencecausing the transcription and expression of the reporter gene. Incertain instances, an agonist of pRB causes an increase in the bindingof pRB to E2f. In certain instances, the increase in binding of pRB andE2f results in a decrease in the transcription and expression of thereporter gene. In certain instances, an antagonist of pRB causes adecrease in the binding of pRB to E2f. In certain instances, thedecrease in binding of pRB and E2f results in a increase in thetranscription and expression of the reporter gene.

In some embodiments, the agent that modulates pRB is an siRNA molecule.In certain instances, the siRNA molecule inhibits the transcription ofan RB1 gene by RNA interference (RNAi). In some embodiments, a doublestranded RNA (dsRNA) molecule with sequences complementary to an RB1mRNA sequence is generated (e.g. by PCR). In some embodiments, a 20-25bp siRNA molecule with sequences complementary to an RB1 mRNA isgenerated. In some embodiments, the 20-25 bp siRNA molecule has 2-5 bpoverhangs on the 3′ end of each strand, and a 5′ phosphate terminus anda 3′ hydroxyl terminus. In some embodiments, the 20-25 bp siRNA moleculehas blunt ends. For techniques for generating RNA sequences seeMolecular Cloning: A Laboratory Manual, second edition (Sambrook et al.,1989) and Molecular Cloning: A Laboratory Manual, third edition(Sambrook and Russel, 2001), jointly referred to herein as “Sambrook”);Current Protocols in Molecular Biology (F. M. Ausubel et al., eds.,1987, including supplements through 2001); Current Protocols in NucleicAcid Chemistry John Wiley & Sons, Inc., New York, 2000) which are herebyincorporated by reference for such disclosure.

In some embodiments, the dsRNA or siRNA molecule is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the inner ear. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the cochlea, the organ of Corti, the vestibular labyrinth, or acombination thereof.

In certain instances, after administration of the dsRNA or siRNAmolecule, cells at the site of administration (e.g. the cells ofcochlea, organ of Corti, and/or the vestibular labyrinth) aretransformed with the dsRNA or siRNA molecule. In certain instancesfollowing transformation, the dsRNA molecule is cleaved into multiplefragments of about 20-25 bp to yield siRNA molecules. In certaininstances, the fragments have about 2 bp overhangs on the 3′ end of eachstrand.

In certain instances, an siRNA molecule is divided into two strands (theguide strand and the anti-guide strand) by an RNA-induced SilencingComplex (RISC). In certain instances, the guide strand is incorporatedinto the catalytic component of the RISC (i.e. argonaute). In certaininstances, the guide strand binds to a complementary RB1 mRNA sequence.In certain instances, the RISC cleaves the RB1 mRNA. In certaininstances, the expression of the RB1 gene is down-regulated.

In some embodiments, a sequence complementary to RB1 mRNA isincorporated into a vector. In some embodiments, the sequence is placedbetween two promoters. In some embodiments, the promoters are orientatedin opposite directions. In some embodiments, the vector is contactedwith a cell. In certain instances, a cell is transformed with thevector. In certain instances following transformation, sense andanti-sense strands of the sequence are generated. In certain instances,the sense and anti-sense strands hybridize to form a dsRNA moleculewhich is cleaved into siRNA molecules. In certain instances, the strandshybridize to form an siRNA molecule. In some embodiments, the vector isa plasmid (e.g. pSUPER; pSUPER.neo; pSUPER.neo+gfp).

In some embodiments, the vector is incorporated into acontrolled-release auris-acceptable microsphere or microparticle,hydrogel, liposome, or thermoreversible gel. In some embodiments, theauris-acceptable microsphere, hydrogel, liposome, paint, foam, in situforming spongy material, nanocapsule or nanosphere or thermoreversiblegel is injected into the inner ear. In some embodiments, theauris-acceptable microsphere or microparticle, hydrogel, liposome, orthermoreversible gel. In some embodiments, the auris-acceptablemicrosphere, hydrogel, liposome, paint, foam, in situ forming spongymaterial, nanocapsule or nanosphere or thermoreversible gel is injectedinto the cochlea, the organ of Corti, the vestibular labyrinth, or acombination thereof.

Thyroid Hormone Receptor Modulation

Contemplated for use with the formulations disclosed herein are agentsthat reduce or reverse the degeneration of neurons and/or hair cells ofthe auris, and/or promote the growth of neurons and/or hair cells of theauris, and agents for treating or ameliorating hearing loss or reductionresulting from destroyed, stunted, malfunctioning, damaged, fragile ormissing hairs in the inner ear. Accordingly, some embodimentsincorporate the use of agents that modulate Thyroid Hormone (TH)receptors. The TH receptors are a family of nuclear hormone receptors.The family includes, but is not limited to TRα1 and TRβ. In certaininstances, TRβ knock-out mice demonstrate a decreased responsiveness toauditory stimuli, and a decrease in K+ current in hair cells.

In some embodiments, the agent that modulates one or more of the THreceptors is an agonist of the one or more TH receptors. In someembodiments, the agonist of one or more of the TH receptors is T3(3,5,3′-triiodo-L-thyronine); KB-141(3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid); GC-1(3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)-phenoxy acetic acid);GC-24 (3,5-dimethyl-4-(4′-hydroxy-3′-benzyl)benzylphenoxyacetic acid);sobetirome (QRX-431); 4-OH-PCB106(4-OH-2′,3,3′,4′,5′-pentachlorobiphenyl); MB07811((2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4-hydroxy-3-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane);MB07344(3,5-dimethyl-4-(4-hydroxy-3-isopropylbenzyl)phenoxy)methylphosphonicacid); and combinations thereof. In certain instances, KB-141; GC-1;sobetirome; and GC-24 are selective for TRβ.

TRPV Modulation

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and hair cells, and agents fortreating or ameliorating hearing loss or reduction resulting fromdestroyed, stunted, malfunctioning, damaged, fragile or missing hairs inthe inner ear. Accordingly, some embodiments incorporate the use ofagents that modulate TRPV receptors. The TRPV (Transient ReceptorPotential Channel Vanilloid) receptors are a family of non-selective ionchannels permeable to calcium, amongst other ions. There are six membersof the family: TRPV1-6. In certain instances, following treatment withkanamycin, TRPV 1 is upregulated. Additionally, in certain instances,antagonism of the TRPV 4 receptor makes mice vulnerable to acoustictrauma. Further, in certain instances, capsaicin, an agonist of TRPV 1,prevents hyperlocomotion following an ischemic event.

In some embodiments, the agent that modulates one or more of the TRPVreceptors is an agonist of the one or more TRPV receptors. In someembodiments, the agonist of one or more of the TRPV receptors iscapsaicin, resiniferatoxin, or combinations thereof. In someembodiments, administration of an auris sensory cell modulatingcomposition comprising a TRPV agonist reduces or reverses degenerationof neurons and hair cells.

Sodium Channel Blockers

Contemplated for use with the formulations disclosed herein are agentsthat modulate the degeneration of neurons and hair cells, and agents fortreating or ameliorating hearing loss or reduction resulting fromdestroyed, stunted, malfunctioning, damaged, fragile or missing hairs inthe inner ear. In certain instances, excitotoxicity causes the excessiveopening of Na+ channels. In certain instances, this results in excessNa+ ions entering the neuron. In certain instances, the excess influx ofNa+ ions into the neuron causes the neuron to fire more often. Incertain instances, this increased firing yields a rapid buildup of freeradicals and inflammatory compounds. In certain instances, the freeradicals damage the mitochondria, depleting the cell's energy stores.Further, in certain instances, excess levels of Na+ ions activate excesslevels of enzymes including, but not limited to, phospholipases,endonucleases, and proteases. In certain instances, the over-activationof these enzymes results in damage to the cytoskeleton, plasma membrane,mitochondria, and DNA of the neuron. Accordingly, some embodimentsincorporate the use of agents which antagonize the opening of Na+channels and reduce or reverse auris hair cell death and/or auris haircell damage.

In some embodiments, the Na+ channel blocker is vinpocetine((3a,16a)-Eburnamenine-14-carboxylic acid ethyl ester); sipatrigine(2-(4-Methylpiperazin-1-yl)-5-(2,3,5-trichlorophenyl)-pyrimidin-4-amine);amiloride (3,5-diamino-N-(aminoiminomethyl)-6-chloropyrazinecarbox amidehydrochloride); carbamazepine (5H-dibenzo[b,f]azepine-5-carboxamide);TTX(octahydro-12-(hydroxymethyl)-2-imino-5,9:7,10a-dimethano-10aH-[1,3]dioxocino[6,5-d]pyrimidine-4,7,10,11,12-pentol);RS100642 (1-(2,6-dimethyl-phenoxy)-2-ethylaminopropane hydrochloride);mexiletine ((1-(2,6-dimethylphenoxy)-2-aminopropane hydrochloride));QX-314 (N-(2,6-Dimethylphenylcarbamoylmethyl)triethylammonium bromide);phenytoin (5,5-diphenylimidazolidine-2,4-dione); lamotrigine(6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine); 4030W92(2,4-diamino-5-(2,3-dichlorophenyl)-6-fluoromethylpyrimidine); BW1003C87(5-(2,3,5-trichlorophenyl) pyrimidine-2,4-1.1 ethanesulphonate); QX-222(2-[(2,6-dimethylphenyl)amino]-N,N,N-trimethyl-2-oxoetha niminiumchloride); ambroxol(trans-4-[[(2-Amino-3,5-dibromophenyl)methyl]amino]cyclo hexanolhydrochloride); R56865(N-[1-(4-(4-fluorophenoxy)butyl]-4-piperidinyl-N-methyl-2-benzo-thiazolamine);lubeluzole; ajmaline ((17R,21 alpha)-ajmalan-17,21-diol); procainamide(4-amino-N-(2-diethylaminoethyl)benzamide hydrochloride); flecainide;riluzoleor; or combinations thereof.

Corticosteroids

Contemplated for use with the compositions and formulations disclosedherein are agents that reduce, reverse or delay the degeneration ofneurons and/or hair cells of the auris, and agents for treating orameliorating hearing loss or reduction resulting from destroyed,stunted, malfunctioning, damaged, fragile or missing hairs in the innerear. Accordingly, some embodiments incorporate the use of agents whichprotect otic hair cells from ototoxins. In some embodiments, the agentwhich protects otic hair cells from ototoxins is a steroid. In someembodiments, the steroid which protects otic hair cells from ototoxinsis a corticosteroid. In some embodiments, the corticosteroid istriamicinolone actenoide and/or dexamethasone. In certain instances,triamicinolone actenoide and dexamethasone protect otic hair cells fromdamage caused by the naturally occurring toxin 4-hydroxy-2,3-nonenal(HNE), which is produced in the inner ear in response to oxidativestress. Other corticosteroids include, and are not limited to,21-acetoxypregnenolone, alclometasone, algestone, amcinonide,beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol,clobetasone, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone,medrysone, meprednisone, methylprednisolone, mometasone furoate,paramethasone, prednicarbate, prednisolone, prednisolone25-diethylamino-acetate, prednisolone sodium phosphate, prednisone,prednival, prednylidene, rimexolone, tixocortol, triamcinolone,triamcinolone acetonide, triamcinolone benetonide, or triamcinolonehexacetonide, or phosphate prodrug or ester prodrug thereof.

Stem Cells and Differentiated Auris Sensory Cells

Contemplated for use with the formulations disclosed herein aretransplants of cells that supplement and/or replace the pre-existingneurons and/or hair cells of the auris. In some embodiments, the agentis a stem cell. In some embodiments, the agent is a partially or fullydifferentiated auris sensory cell. In some embodiments, thedifferentiated auris sensory cell is derived from a human donor. In someembodiments, the differentiated auris sensory cell is derived from astem cell, the differentiation of which was induced under artificial(e.g. laboratory) conditions.

Stem cells are cells that possess the capacity to differentiate intomultiple cell types. Totipotent stem cells can differentiate intoembryonic cells or extraembryonic cells. Pluripotent cells candifferentiate into cells of any of endoderm, mesoderm, or ectodermorigin. Multipotent cells can differentiate into closely related cells(e.g. hematopoietic stem cells). Unipotent cells can differentiate intoonly one type of cell, but like other stem cells have the characteristicof self-renewal. In some embodiments, the stem cell is totipotent,pluripotent, multipotent, or unipotent. Further, stem cells can undergomitotic division without themselves differentiating (i.e. self-renewal).

Embryonic stem (ES) cells are stem cells derived from the epiblasttissue of the inner cell mass of a blastocyst or earlier stage embryo.ES cells are pluripotent. In some embodiments, the stem cell is an EScell. Adult stem cells (also known as somatic cells or germline cells)are cells isolated from a developed organism wherein the cells possessthe characteristic of self-renewal, and the ability to differentiateinto multiple cell types. Adult stem cells are pluripotent (for example,stem cells found in umbilical cord blood), multipotent or unipotent. Insome embodiments, the stem cell is an adult stem cell.

In some embodiments, a stem cell and/or a differentiated auris sensorycell is administered in combination with a differentiation stimulatingagent. In some embodiments, the differentiation stimulating agent is agrowth factor. In some embodiments, the growth factor is a neurotrophin(e.g. nerve growth factor (NGF), brain-derived neurotrophic factor(BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), or novelneurotrophin-1 (NNT1). In some embodiments, the growth factor is FGF,EGF, IGF, PGF, or combinations thereof.

In some embodiments, a stem cell and/or a differentiated auris sensorycell is administered to a subject in need thereof as a controlledrelease agent. In some embodiments, a stem cell and/or a differentiatedauris sensory cell is administered to a subject in need thereof as animmediate release agent (e.g. in a cell suspension) in combination witha controlled release auris sensory cell modulating agent. In someembodiments, a controlled release auris sensory cell modulating agent isa vector comprising an Atoh1 or BRN3 gene, an siRNA sequence targetingRB1, a growth factor, or combinations thereof.

In some embodiments, a stem cell and/or a differentiated auris sensorycell is administered to the cochlea or vestibular labyrinth. In someembodiments, a stem cell and/or a differentiated auris sensory cell isadministered by via intratympanic injection, and/or a post-auricularincision. In some embodiments, a stem cell and/or a differentiated aurissensory cell is contacted with the organ of Corti, vestibulocochlearnerve, and/or crista ampullaris.

Immediate Release Agents

In some embodiments, the composition further comprises a modulator ofneuron and/or hair cells of the auris as an immediate release agentwherein the immediate release modulator of neuron and/or hair cells ofthe auris is the same agent as the controlled-release agent, a differentmodulator of neuron and/or hair cells of the auris, an additionaltherapeutic agent, or a combination thereof. In some embodiments, theimmediate release agent is a stem cell, a differentiated auris sensorycell, an immune system cell, a vector carrying a copy of an Atoh1 gene,a vector carrying a copy of a BRN3 gene, an siRNA sequence, an miRNAsequence, or combinations thereof.

Direct Injection

In some embodiments, one of the agents is directly injected into theauris interna, including through the round window membrane, while asecond agent is administered in the auris media, on or in contact withthe round window membrane such that the second agent is in the form of acontrolled release formulation. In one embodiment, thedirectly-administered agent is a stem cell, a differentiated aurissensory cell, an immune system cell, a vector carrying a copy of anAtoh1 gene, a vector carrying a copy of a BRN3 gene, an siRNA sequence,an miRNA sequence, or combinations thereof.

Concentration of Active Agent

In some embodiments, the compositions described herein have aconcentration of active pharmaceutical ingredient between about 0.01% toabout 90%, between about 0.01% to about 50%, between about 0.1% to about70%, between about 0.1% to about 50%, between about 0.1% to about 40%,between about 0.1% to about 30%, between about 0.1% to about 20%,between about 0.1% to about 10%, or between about 0.1% to about 5%, ofthe active ingredient, or pharmaceutically acceptable prodrug or saltthereof, by weight of the composition. In some embodiments, thecompositions described herein have a concentration of activepharmaceutical agent between about 1% to about 50%, between about 5% toabout 50%, between about 10% to about 40%, or between about 10% to about30%, of the active ingredient, or pharmaceutically acceptable prodrug orsalt thereof, by weight of the composition. In some embodiments,formulations described herein comprise about 70% by weight of an aurissensory cell modulating agent by weight of the formulation. In someembodiments, formulations described herein comprise about 60% by weightof an auris sensory cell modulating agent by weight of the formulation.In some embodiments, formulations described herein comprise about 50% byweight of an auris sensory cell modulating agent by weight of theformulation. In some embodiments, formulations described herein compriseabout 40% by weight of an auris sensory cell modulating agent by weightof the formulation. In some embodiments, formulations described hereincomprise about 30% by weight of an auris sensory cell modulating agentby weight of the formulation. In some embodiments, formulationsdescribed herein comprise about 20% by weight of an auris sensory cellmodulating agent by weight of the formulation. In some embodiments,formulations described herein comprise about 15% by weight of an aurissensory cell modulating agent, or pharmaceutically acceptable prodrug orsalt thereof, by weight of the formulation. In some embodiments,formulations described herein comprise about 10% by weight of an aurissensory cell modulating agent by weight of the formulation. In someembodiments, formulations described herein comprise about 5% by weightof an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, formulations described herein comprise about 2.5% byweight of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, formulations described herein comprise about 1% byweight of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, formulations described herein comprise about 0.5% byweight of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, formulations described herein comprise about 0.1% byweight of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, formulations described herein comprise about 0.01% byweight of an auris sensory cell modulating agent, or pharmaceuticallyacceptable prodrug or salt thereof, by weight of the formulation. Insome embodiments, the formulations described herein have a concentrationof active pharmaceutical ingredient, or pharmaceutically acceptableprodrug or salt thereof, between about 0.1 to about 70 mg/mL, betweenabout 0.5 mg/mL to about 70 mg/mL, between about 0.5 mg/mL to about 50mg/mL, between about 0.5 mg/mL to about 20 mg/mL, between about 1 mg toabout 70 mg/mL, between about 1 mg to about 50 mg/mL, between about 1mg/mL and about 20 mg/mL, between about 1 mg/mL to about 10 mg/mL, orbetween about 1 mg/mL to about 5 mg/mL, of the active agent, orpharmaceutically acceptable prodrug or salt thereof, by volume of theformulation.

Combination Therapy

In some embodiments, any composition or device described hereincomprises one or more active agents and/or a second therapeutic agentincluding but not limited to anti-emetic agents, antimicrobial agents,antioxidants, anti-septic agents or the like.

Anti-Emetic Agents

Anti-Emetic agents are optionally used in combination with theformulations disclosed herein. Anti-emetic agents include promethazine,prochlorperazine, trimethobenzamide, and triethylperazine. Otheranti-emetic agents include 5HT3 antagonists such as dolasetron,granisetron, ondansetron, tropisetron, and palonosetron; andneuroleptics such as droperidol. Further anti-emetic agents includeantihistamines, such as meclizine; phenothiazines such as perphenazine,and thiethylperazine; dopamine antagonists, including domperidone,properidol, haloperidol, chlorpromazine, promethazine, prochlorperazine,metoclopramide and combinations thereof; cannabinoids, includingdronabinol, nabilone, sativex, and combinations thereof;anticholinergics, including scopolamine; and steroids, includingdexamethasone; trimethobenzamine, emetrol, propofol, muscimol, andcombinations thereof.

Antimicrobial Agents

Antimicrobial agents are also contemplated as useful with theformulations disclosed herein. Antimicrobial agents include agents thatact to inhibit or eradicate microbes, including bacteria, fungi orparasites. Specific antimicrobial agents may be used to combat specificmicrobes. Accordingly, a skilled practitioner would know whichantimicrobial agent would be relevant or useful depending on the microbeidentified, or the symptoms displayed. Antimicrobial agents includeantibiotics, antiviral agents, antifungal agents, and antiparasiticagents.

Antibiotics may include amikacin, gentamicin, kanamycin, neomycin,netilmicin, streptomycin, tobramycin, paromomycin, geldanmycin,herbimycin, loracarbef, ertapenem, doripenem, imipenem, cilastatin,meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor,cefamandole, cefoxitin, defprozil, cefuroxime, cefixime, cefdinir,cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime,ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole,teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin,spectinomycin, aztreonam, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,meticillin, nafcillin, oxacillin, penicillin, piperacillin, ticarcillan,bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovfloxacin, mafenide, prontosil, sulfacetamide,sulfamethizole, sulfanimilimde, sulfsalazine, sulfsioxazole,trimethoprim, demeclocycline, doxycycline, minocycline, oxtetracycline,tetracycline, arsphenamine, chloramphenicol, clindamycin, lincomycin,ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid,linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin,pyrazinamide, quinuspristin/dalfopristin, rifampin, tinidazole, andcombinations thereof.

Antiviral agents may include acyclovir, famciclovir and valacyclovir.Other antiviral agents include abacavir, aciclovir, adfovir, amantadine,amprenavir, arbidol., atazanavir, artipla, brivudine, cidofovir,combivir, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,fomvirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, gardasil,ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine,integrase inhibitors, interferons, including interferon type III,interferon type II, interferon type I, lamivudine, lopinavir, loviride,MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir,nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril,podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors,ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir,tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine,truvada, valganciclovir, vicriviroc, vidarabine, viramidine,zalcitabine, zanamivir, zidovudine, and combinations thereof.

Antifungal agents may include amrolfine, utenafine, naftifine,terbinafine, flucytosine, fluconazole, itraconazole, ketoconazole,posaconazole, ravuconazole, voriconazole, clotrimazole, econazole,miconazole, oxiconazole, sulconazole, terconazole, tioconazole,nikkomycin Z, caspofungin, micafungin, anidulafungin, amphotericin B,liposomal nystastin, pimaricin, griseofulvin, ciclopirox olamine,haloprogin, tolnaftate, undecylenate, and combinations thereof.Antiparasitic agents may include amitraz, amoscanate, avermectin,carbadox, diethylcarbamizine, dimetridazole, diminazene, ivermectin,macrofilaricide, malathion, mitaban, oxaminiquine, permethrin,praziquantel, prantel pamoate, selamectin, sodium stibogluconate,thiabendazole, and combinations thereof.

Antioxidants

Antioxidants are optionally used in combination with the compositionsdescribed herein. Antioxidants are also contemplated as being usefulwith the formulations disclosed herein as agents that modulate thedegeneration of neurons and/or hair cells of the auris. Accordingly,some embodiments incorporate the use of antioxidants. In someembodiments, the antioxidant is vitamin C, N-acetylcysteine, vitamin E,Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one (also called PZ 51 orDR3305), L-methionine, Idebenone(2-(10-hydroxydecyl)-5,6-dimethoxy-3-methyl-cyclohexa-2,5-diene-1,4-dione).In some embodiments, antioxidants are trophic agents and promote growthof healthy cells.

Anti-Septic Agents

Anti-septic agents are optionally used in combination with thecompositions described herein. Anti-septic agents are also contemplatedas useful with the formulations disclosed herein. Anti-septic agentsinclude, but are not limited to, acetic acid, boric acid, gentianviolet, hydrogen peroxide, carbamide peroxide, chlorhexidine, saline,mercurochrome, povidone iodine, polyhyroxine iodine, cresylate andaluminum acetate, and mixtures thereof.

Other agents are optionally used in any composition or device describedherein. In some embodiments, a monoamine oxidase inhibitor (e.g.,Rasagiline, R(+)—N-propargyl-1-aminoindan) is used in any composition ordevice described herein. In some embodiments, an adenosine antagonist(e.g., R—N6-Phenylisopropyl adenosine, 1-2-oxothiazolidine-4-carboxylicacid (Procysteine)) is used in any composition or device describedherein. Any combination of active agents and/or second therapeutic agentis compatible with the compositions described herein.

Otic Surgery and Implants

In some embodiments, the pharmaceutical formulations, compositions ordevices described herein are used in combination with (e.g.,implantation, short-term use, long-term use, or removal of) implants(e.g., cochlear implants). As used herein, implants includeauris-interna or auris-media medical devices, examples of which includecochlear implants, hearing sparing devices, hearing-improvement devices,short electrodes, micro-prostheses or piston-like prostheses; needles;stem cell transplants; drug delivery devices; any cell-basedtherapeutic; or the like. In some instances, the implants are used inconjunction with a patient experiencing hearing loss. In some instances,the hearing loss is present at birth. In some instances, the hearingloss is associated with conditions such as AIED, bacterial meningitis orthe like that lead to osteoneogenesis and/or nerve damage with rapidobliteration of cochlear structures and profound hearing loss.

In some instances, an implant is an immune cell or a stem celltransplant in the ear. In some instances, an implant is a smallelectronic device that has an external portion placed behind the ear,and a second portion that is surgically placed under the skin that helpsprovide a sense of sound to a person who is profoundly deaf or severelyhard-of-hearing. By way of example, such cochlear medical deviceimplants bypass damaged portions of the ear and directly stimulate theauditory nerve. In some instances cochlear implants are used in singlesided deafness. In some instances cochlear implants are used fordeafness in both ears.

In some embodiments, administration of an auris sensory cell modulatorcomposition or device described herein in combination with an oticintervention (e.g., an intratympanic injection, a stapedectomy, amedical device implant or a cell-based transplant) delays or preventscollateral damage to auris structures, e.g., irritation, cell damage,cell death, osteoneogenesis and/or further neuronal degeneration, causedby the external otic intervention (e.g., installation of an externaldevice and/or cells in the ear). In some embodiments, administration ofan auris sensory cell modulator composition or device described hereinin combination with an implant allows for a more effective restorationof hearing loss compared to an implant alone.

In some embodiments, administration of an auris sensory cell modulatorcomposition or device described herein reduces damage to cochlearstructures caused by underlying conditions (e.g., bacterial meningitis,autoimmune ear disease (AIED)) allowing for successful cochlear deviceimplantation. In some embodiments, administration of a composition ordevice described herein, in conjunction with otic surgery, medicaldevice implantation and/or cell transplantation, reduces or preventscell damage and/or death (e.g., auris sensory hair cell death and/ordamage) associated with otic surgery, medical device implantation and/orcell transplantation.

In some embodiments, administration of an auris sensory cell modulatorcomposition or device described herein (e.g., a composition or devicecomprising a growth factor) in conjunction with a cochlear implant orstem cell transplant has a trophic effect (e.g., promotes healthy growthof cells and/or healing of tissue in the area of an implant ortransplant). In some embodiments, a trophic effect is desirable duringotic surgery or during intratympanic injection procedures. In someembodiments, a trophic effect is desirable after installation of amedical device or after a cell transplant. In some of such embodiments,the auris sensory cell modulator compositions or devices describedherein are administered via direct cochlear injection, through achochleostomy or via deposition on the round window.

In some embodiments, administration of an anti-inflammatory orimmunosuppressant composition (e.g., a composition comprising animmunosuppressant such as a corticosteroid) reduces inflammation and/orinfections associated with otic surgery, implantation of a medicaldevice or a cell transplant. In some instances, perfusion of a surgicalarea with an auris sensory cell modulator formulation described hereinreduces or eliminates post-surgical and/or post-implantationcomplications (e.g., inflammation, hair cell damage, neuronaldegeneration, osteoneogenesis or the like). In some instances, perfusionof a surgical area with a formulation described herein reducespost-surgery or post-implantation recuperation time. In someembodiments, a medical device is coated with a composition describedherein prior to implantation in the ear.

In one aspect, the formulations described herein, and modes ofadministration thereof, are applicable to methods of direct perfusion ofthe inner ear compartments. Thus, the formulations described herein areuseful in combination with otic interventions. In some embodiments, anotic intervention is an implantation procedure (e.g., implantation of ahearing device in the cochlea). In some embodiments, an oticintervention is a surgical procedure including, by way of non-limitingexamples, cochleostomy, labyrinthotomy, mastoidectomy, stapedectomy,stapedotomy, endolymphatic sacculotomy, tympanostomy or the like. Insome embodiments, the inner ear compartments are perfused with aformulation described herein prior to otic intervention, during oticintervention, or after otic intervention, or a combination thereof.

In some embodiments, when perfusion is carried out in combination withotic intervention, the auris sensory cell compositions are immediaterelease compositions. In some of such embodiments, the immediate releaseformulations described herein are non-thickened compositions and aresubstantially free of extended release components (e.g., gellingcomponents such as polyoxyethylene-polyoxypropylene copolymers). In someof such embodiments, the compositions contain less than 5% of theextended release components (e.g., gelling components such aspolyoxyethylene-polyoxypropylene triblock copolymers) by weight of theformulation. In some of such embodiments, the compositions contain lessthan 2% of the extended release components (e.g., gelling componentssuch as polyoxyethylene-polyoxypropylene triblock copolymers) by weightof the formulation. In some of such embodiments, the compositionscontain less than 1% of the extended release components (e.g., gellingcomponents such as polyoxyethylene-polyoxypropylene triblock copolymers)by weight of the formulation. In some of such embodiments, a compositiondescribed herein that is used for perfusion of a surgical area containssubstantially no gelling component and is an immediate releasecomposition.

In other embodiments, a composition described herein is administeredafter an otic intervention (e.g., after implantation of a medical deviceor a cell-based therapeutic). In some of such embodiments, a compositiondescribed herein that is administered after the otic intervention is anintermediate release or extended release composition and containsgelling components as described herein.

Presented below (Table 1) are examples of active agents contemplated foruse with the formulations and devices disclosed herein. One or moreactive agents are used in any of the formulations or devices describedherein.

Active Agents (including pharmaceutically acceptable salts of theseactive agents) for use with the Formulations Disclosed Herein

TABLE 1 Auris Condition Therapeutic Agent Benign ParoxysmalDiphenhydramine Positional Vertigo Benign Paroxysmal LorazepamPositional Vertigo Benign Paroxysmal Meclizine Positional Vertigo BenignParoxysmal Oldansetron Positional Vertigo Hearing Loss Estrogen VertigoDNQX Vertigo 6-OH Dopamine Tinnitus (S)-Ketamine Hearing Loss Estrogenand progesterone (E + P) Hearing Loss Folic acid Hearing Loss LactatedRinger's with 0.03% Ofloxacin Hearing Loss Methotrexate Hearing LossN-acetyl cysteine Meniere's Disease Betahistine Meniere's DiseaseSildenafil Meniere's Disease conivaptan Middle Ear EffusionPneumonococcal vaccine Otitis Externa Diclofenac sodium; dexotc OtitisExterna, Acute AL-15469A/AL-38905 Otitis Media Amoxicillin/clavulanateOtitis Media Dornase alfa Otitis Media Echinacea purpurea Otitis MediaFaropenem medoxomil Otitis Media Levofloxacin Otitis Media PNCRM9 OtitisMedia Pneumococcal vaccine Otitis Media Telithromycin Otitis Media ZmaxOtitis Media with Lansoprazole Effusion Otitis Media, Acute AL-15469A;AL-38905 Otitis Media, Acute Amoxicillin Otitis Media, AcuteAmoxicillin-clavulanate Otitis Media, Acute Azithromycin Otitis Media,Acute Azithromycin SR Otitis Media, Acute Cefdinir Otitis Media, AcuteHyland's earache drops Otitis Media, Acute Montelukast Otitis Media,Acute Pneumonococcal vaccine Otitis Media, Acute AL-15469A/AL38905 withTypanostomy Tubes Otitis Media, Chronic Sulfamethoxazole- trimethoprimOtitis Media, Azithromycin Suppurative Otitis Media, TelithromycinSuppurative Otosclerosis Acetylcysteine Ototoxicity Aspirin TinnitusAcamprosate Tinnitus Gabapentin Tinnitus Modafinil Tinnitus NeramexaneTinnitus Neramexane mesylate Tinnitus Piribedil Tinnitus VardenafilTinnitus Vestipitant + Paroxetine Tinnitus Vestiplitant Tinnitus Zincsulfate

General Methods of Sterilization

Provided herein are otic compositions that ameliorate or lessen oticdisorders described herein. Further provided herein are methodscomprising the administration of said otic compositions. In someembodiments, the compositions or devices are sterilized. Included withinthe embodiments disclosed herein are means and processes forsterilization of a pharmaceutical composition or device disclosed hereinfor use in humans. The goal is to provide a safe pharmaceutical product,relatively free of infection causing micro-organisms. The U.S. Food andDrug Administration has provided regulatory guidance in the publication“Guidance for Industry: Sterile Drug Products Produced by AsepticProcessing” available at: http://www.fda.gov/cder/guidance/5882fnl.htm,which is incorporated herein by reference in its entirety.

As used herein, sterilization means a process used to destroy or removemicroorganisms that are present in a product or packaging. Any suitablemethod available for sterilization of objects and compositions is used.Available methods for the inactivation of microorganisms include, butare not limited to, the application of extreme heat, lethal chemicals,or gamma radiation. In some embodiments is a process for the preparationof an otic therapeutic formulation comprising subjecting the formulationto a sterilization method selected from heat sterilization, chemicalsterilization, radiation sterilization or filtration sterilization. Themethod used depends largely upon the nature of the device or compositionto be sterilized. Detailed descriptions of many methods of sterilizationare given in Chapter 40 of Remington: The Science and Practice ofPharmacy published by Lippincott, Williams & Wilkins, and isincorporated by reference with respect to this subject matter.

Sterilization by Heat

Many methods are available for sterilization by the application ofextreme heat. One method is through the use of a saturated steamautoclave. In this method, saturated steam at a temperature of at least121° C. is allowed to contact the object to be sterilized. The transferof heat is either directly to the microorganism, in the case of anobject to be sterilized, or indirectly to the microorganism by heatingthe bulk of an aqueous solution to be sterilized. This method is widelypracticed as it allows flexibility, safety and economy in thesterilization process.

Dry heat sterilization is a method which is used to kill microorganismsand perform depyrogenation at elevated temperatures. This process takesplace in an apparatus suitable for heating HEPA-filteredmicroorganism-free air to temperatures of at least 130-180° C. for thesterilization process and to temperatures of at least 230-250° C. forthe depyrogenation process. Water to reconstitute concentrated orpowdered formulations is also sterilized by autoclave. In someembodiments, the formulations described herein comprise micronized aurissensory cell modulating agents (e.g., micro-ketamine powder) that aresterilized by dry heating, e.g., heating for about 7-11 hours atinternal powder temperatures of 130-140° C., or for 1-2 hours atinternal temperatures of 150-180° C.

Chemical Sterilization

Chemical sterilization methods are an alternative for products that donot withstand the extremes of heat sterilization. In this method, avariety of gases and vapors with germicidal properties, such as ethyleneoxide, chlorine dioxide, formaldehyde or ozone are used as theanti-apoptotic agents. The germicidal activity of ethylene oxide, forexample, arises from its ability to serve as a reactive alkylatingagent. Thus, the sterilization process requires the ethylene oxidevapors to make direct contact with the product to be sterilized.

Radiation Sterilization

One advantage of radiation sterilization is the ability to sterilizemany types of products without heat degradation or other damage. Theradiation commonly employed is beta radiation or alternatively, gammaradiation from a ⁶⁰Co source. The penetrating ability of gamma radiationallows its use in the sterilization of many product types, includingsolutions, compositions and heterogeneous mixtures. The germicidaleffects of irradiation arise from the interaction of gamma radiationwith biological macromolecules. This interaction generates chargedspecies and free radicals. Subsequent chemical reactions, such asrearrangements and cross-linking processes, result in the loss of normalfunction for these biological macromolecules. The formulations describedherein are also optionally sterilized using beta irradiation.

Filtration

Filtration sterilization is a method used to remove but not destroymicroorganisms from solutions. Membrane filters are used to filterheat-sensitive solutions. Such filters are thin, strong, homogenouspolymers of mixed cellulosic esters (MCE), polyvinylidene fluoride (PVF;also known as PVDF), or polytetrafluoroethylene (PTFE) and have poresizes ranging from 0.1 to 0.22 μm. Solutions of various characteristicsare optionally filtered using different filter membranes. For example,PVF and PTFE membranes are well suited to filtering organic solventswhile aqueous solutions are filtered through PVF or MCE membranes.Filter apparatus are available for use on many scales ranging from thesingle point-of-use disposable filter attached to a syringe up tocommercial scale filters for use in manufacturing plants. The membranefilters are sterilized by autoclave or chemical sterilization.Validation of membrane filtration systems is performed followingstandardized protocols (Microbiological Evaluation of Filters forSterilizing Liquids, Vol 4, No. 3. Washington, D.C: Health IndustryManufacturers Association, 1981) and involve challenging the membranefilter with a known quantity (ca. 10^(7/)cm²) of unusually smallmicroorganisms, such as Brevundimonas diminuta (ATCC 19146).

Pharmaceutical compositions are optionally sterilized by passing throughmembrane filters. Formulations comprising nanoparticles (U.S. Pat. No.6,139,870) or multilamellar vesicles (Richard et al., InternationalJournal of Pharmaceutics (2006), 312(1-2): 144-50) are amenable tosterilization by filtration through 0.22 μm filters without destroyingtheir organized structure.

In some embodiments, the methods disclosed herein comprise sterilizingthe formulation (or components thereof) by means of filtrationsterilization. In another embodiment the auris-acceptable otictherapeutic agent formulation comprises a particle wherein the particleformulation is suitable for filtration sterilization. In a furtherembodiment said particle formulation comprises particles of less than300 nm in size, of less than 200 nm in size, of less than 100 nm insize. In another embodiment the auris-acceptable formulation comprises aparticle formulation wherein the sterility of the particle is ensured bysterile filtration of the precursor component solutions. In anotherembodiment the auris-acceptable formulation comprises a particleformulation wherein the sterility of the particle formulation is ensuredby low temperature sterile filtration. In a further embodiment, lowtemperature sterile filtration is carried out at a temperature between 0and 30° C., between 0 and 20° C., between 0 and 10° C., between 10 and20° C., or between 20 and 30° C.

In another embodiment is a process for the preparation of anauris-acceptable particle formulation comprising: filtering the aqueoussolution containing the particle formulation at low temperature througha sterilization filter; lyophilizing the sterile solution; andreconstituting the particle formulation with sterile water prior toadministration. In some embodiments, a formulation described herein ismanufactured as a suspension in a single vial formulation containing themicronized active pharmaceutical ingredient. A single vial formulationis prepared by aseptically mixing a sterile poloxamer solution withsterile micronized active ingredient (e.g., ketamine) and transferringthe formulation to sterile pharmaceutical containers. In someembodiments, a single vial containing a formulation described herein asa suspension is resuspended before dispensing and/or administration.

In specific embodiments, filtration and/or filling procedures arecarried out at about 5° C. below the gel temperature (Tgel) of aformulation described herein and with viscosity below a theoreticalvalue of 100 cP to allow for filtration in a reasonable time using aperistaltic pump.

In another embodiment the auris-acceptable otic therapeutic agentformulation comprises a nanoparticle formulation wherein thenanoparticle formulation is suitable for filtration sterilization. In afurther embodiment the nanoparticle formulation comprises nanoparticlesof less than 300 nm in size, of less than 200 nm in size, or of lessthan 100 nm in size. In another embodiment the auris-acceptableformulation comprises a microsphere formulation wherein the sterility ofthe microsphere is ensured by sterile filtration of the precursororganic solution and aqueous solutions. In another embodiment theauris-acceptable formulation comprises a thermoreversible gelformulation wherein the sterility of the gel formulation is ensured bylow temperature sterile filtration. In a further embodiment, the lowtemperature sterile filtration occurs at a temperature between 0 and 30°C., or between 0 and 20° C., or between 0 and 10° C., or between 10 and20° C., or between 20 and 30° C. In another embodiment is a process forthe preparation of an auris-acceptable thermoreversible gel formulationcomprising: filtering the aqueous solution containing thethermoreversible gel components at low temperature through asterilization filter; lyophilizing the sterile solution; andreconstituting the thermoreversible gel formulation with sterile waterprior to administration.

In certain embodiments, the active ingredients are dissolved in asuitable vehicle (e.g. a buffer) and sterilized separately (e.g. by heattreatment, filtration, gamma radiation). In some instances, the activeingredients are sterilized separately in a dry state. In some instances,the active ingredients are sterilized as a suspension or as a colloidalsuspension. The remaining excipients (e.g., fluid gel components presentin auris formulations) are sterilized in a separate step by a suitablemethod (e.g. filtration and/or irradiation of a cooled mixture ofexcipients); the two solutions that are separately sterilized are thenmixed aseptically to provide a final auris formulation. In someinstances, the final aseptic mixing is performed just prior toadministration of a formulation described herein.

In some instances, conventionally used methods of sterilization (e.g.,heat treatment (e.g., in an autoclave), gamma irradiation, filtration)lead to irreversible degradation of polymeric components (e.g.,thermosetting, gelling or mucoadhesive polymer components) and/or theactive agent in the formulation. In some instances, sterilization of anauris formulation by filtration through membranes (e.g., 0.2 μMmembranes) is not possible if the formulation comprises thixotropicpolymers that gel during the process of filtration.

Accordingly, provided herein are methods for sterilization of aurisformulations that prevent degradation of polymeric components (e.g.,thermosetting and/or gelling and/or mucoadhesive polymer components)and/or the active agent during the process of sterilization. In someembodiments, degradation of the active agent (e.g., any therapeutic oticagent described herein) is reduced or eliminated through the use ofspecific pH ranges for buffer components and specific proportions ofgelling agents in the formulations. In some embodiments, the choice ofan appropriate gelling agent and/or thermosetting polymer allows forsterilization of formulations described herein by filtration. In someembodiments, the use of an appropriate thermosetting polymer and anappropriate copolymer (e.g., a gelling agent) in combination with aspecific pH range for the formulation allows for high temperaturesterilization of formulations described with substantially nodegradation of the therapeutic agent or the polymeric excipients. Anadvantage of the methods of sterilization provided herein is that, incertain instances, the formulations are subjected to terminalsterilization via autoclaving without any loss of the active agentand/or excipients and/or polymeric components during the sterilizationstep and are rendered substantially free of microbes and/or pyrogens.

Microorganisms

Provided herein are auris-acceptable compositions or devices thatameliorate or lessen otic disorders described herein. Further providedherein are methods comprising the administration of said oticcompositions. In some embodiments, the compositions or devices aresubstantially free of microorganisms. Acceptable bioburden or sterilitylevels are based on applicable standards that define therapeuticallyacceptable compositions, including but not limited to United StatesPharmacopeia Chapters <1111> et seq. For example, acceptable sterility(e.g., bioburden) levels include about 10 colony forming units (cfu) pergram of formulation, about 50 cfu per gram of formulation, about 100 cfuper gram of formulation, about 500 cfu per gram of formulation or about1000 cfu per gram of formulation. In some embodiments, acceptablebioburden levels or sterility for formulations include less than 10cfu/mL, less that 50 cfu/mL, less than 500 cfu/mL or less than 1000cfu/mL microbial agents. In addition, acceptable bioburden levels orsterility include the exclusion of specified objectionablemicrobiological agents. By way of example, specified objectionablemicrobiological agents include but are not limited to Escherichia coli(E. coli), Salmonella sp., Pseudomonas aeruginosa (P. aeruginosa) and/orother specific microbial agents.

Sterility of the auris-acceptable otic therapeutic agent formulation isconfirmed through a sterility assurance program in accordance withUnited States Pharmacopeia Chapters <61>, <62> and <71>. A key componentof the sterility assurance quality control, quality assurance andvalidation process is the method of sterility testing. Sterilitytesting, by way of example only, is performed by two methods. The firstis direct inoculation wherein a sample of the composition to be testedis added to growth medium and incubated for a period of time up to 21days. Turbidity of the growth medium indicates contamination. Drawbacksto this method include the small sampling size of bulk materials whichreduces sensitivity, and detection of microorganism growth based on avisual observation. An alternative method is membrane filtrationsterility testing. In this method, a volume of product is passed througha small membrane filter paper. The filter paper is then placed intomedia to promote the growth of microorganisms. This method has theadvantage of greater sensitivity as the entire bulk product is sampled.The commercially available Millipore Steritest sterility testing systemis optionally used for determinations by membrane filtration sterilitytesting. For the filtration testing of creams or ointments Steritestfilter system No. TLHVSL210 are used. For the filtration testing ofemulsions or viscous products Steritest filter system No. TLAREM210 orTDAREM210 are used. For the filtration testing of pre-filled syringesSteritest filter system No. TTHASY210 are used. For the filtrationtesting of material dispensed as an aerosol or foam Steritest filtersystem No. TTHVA210 are used. For the filtration testing of solublepowders in ampoules or vials Steritest filter system No. TTHADA210 orTTHADV210 are used.

Testing for E. coli and Salmonella includes the use of lactose brothsincubated at 30-35° C. for 24-72 hours, incubation in MacConkey and/orEMB agars for 18-24 hours, and/or the use of Rappaport medium. Testingfor the detection of P. aeruginosa includes the use of NAC agar. UnitedStates Pharmacopeia Chapter <62> further enumerates testing proceduresfor specified objectionable microorganisms.

In certain embodiments, any controlled release formulation describedherein has less than about 60 colony forming units (CFU), less thanabout 50 colony forming units, less than about 40 colony forming units,or less than about 30 colony forming units of microbial agents per gramof formulation. In certain embodiments, the otic formulations describedherein are formulated to be isotonic with the endolymph and/or theperilymph.

Endotoxins

Provided herein are otic compositions that ameliorate or lessen oticdisorders described herein. Further provided herein are methodscomprising the administration of said otic compositions. In someembodiments, the compositions or devices are substantially free ofendotoxins. An additional aspect of the sterilization process is theremoval of by-products from the killing of microorganisms (hereinafter,“Product”). The process of depyrogenation removes pyrogens from thesample. Pyrogens are endotoxins or exotoxins which induce an immuneresponse. An example of an endotoxin is the lipopolysaccharide (LPS)molecule found in the cell wall of gram-negative bacteria. Whilesterilization procedures such as autoclaving or treatment with ethyleneoxide kill the bacteria, the LPS residue induces a proinflammatoryimmune response, such as septic shock. Because the molecular size ofendotoxins can vary widely, the presence of endotoxins is expressed in“endotoxin units” (EU). One EU is equivalent to 100 picograms of E. coliLPS. Humans can develop a response to as little as 5 EU/kg of bodyweight. The bioburden (e.g., microbial limit) and/or sterility (e.g.,endotoxin level) is expressed in any units as recognized in the art. Incertain embodiments, otic compositions described herein contain lowerendotoxin levels (e.g. <4 EU/kg of body weight of a subject) whencompared to conventionally acceptable endotoxin levels (e.g., 5 EU/kg ofbody weight of a subject). In some embodiments, the auris-acceptableotic therapeutic agent formulation has less than about 5 EU/kg of bodyweight of a subject. In other embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 4 EU/kg of body weightof a subject. In additional embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 3 EU/kg of body weightof a subject. In additional embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 2 EU/kg of body weightof a subject.

In some embodiments, the auris-acceptable otic therapeutic agentformulation or device has less than about 5 EU/kg of formulation. Inother embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 4 EU/kg of formulation. In additionalembodiments, the auris-acceptable otic therapeutic agent formulation hasless than about 3 EU/kg of formulation. In some embodiments, theauris-acceptable otic therapeutic agent formulation has less than about5 EU/kg Product. In other embodiments, the auris-acceptable otictherapeutic agent formulation has less than about 1 EU/kg Product. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 0.2 EU/kg Product. In some embodiments,the auris-acceptable otic therapeutic agent formulation has less thanabout 5 EU/g of unit or Product. In other embodiments, theauris-acceptable otic therapeutic agent formulation has less than about4 EU/g of unit or Product. In additional embodiments, theauris-acceptable otic therapeutic agent formulation has less than about3 EU/g of unit or Product. In some embodiments, the auris-acceptableotic therapeutic agent formulation has less than about 5 EU/mg of unitor Product. In other embodiments, the auris-acceptable otic therapeuticagent formulation has less than about 4 EU/mg of unit or Product. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 3 EU/mg of unit or Product. In certainembodiments, otic compositions described herein contain from about 1 toabout 5 EU/mL of formulation. In certain embodiments, otic compositionsdescribed herein contain from about 2 to about 5 EU/mL of formulation,from about 3 to about 5 EU/mL of formulation, or from about 4 to about 5EU/mL of formulation.

In certain embodiments, otic compositions or devices described hereincontain lower endotoxin levels (e.g. <0.5 EU/mL of formulation) whencompared to conventionally acceptable endotoxin levels (e.g., 0.5 EU/mLof formulation). In some embodiments, the auris-acceptable otictherapeutic agent formulation or device has less than about 0.5 EU/mL offormulation. In other embodiments, the auris-acceptable otic therapeuticagent formulation has less than about 0.4 EU/mL of formulation. Inadditional embodiments, the auris-acceptable otic therapeutic agentformulation has less than about 0.2 EU/mL of formulation.

Pyrogen detection, by way of example only, is performed by severalmethods. Suitable tests for sterility include tests described in UnitedStates Pharmacopoeia (USP) <71> Sterility Tests (23rd edition, 1995).The rabbit pyrogen test and the Limulus amebocyte lysate test are bothspecified in the United States Pharmacopeia Chapters <85> and <151>(USP23/NF 18, Biological Tests, The United States PharmacopeialConvention, Rockville, Md., 1995). Alternative pyrogen assays have beendeveloped based upon the monocyte activation-cytokine assay. Uniformcell lines suitable for quality control applications have been developedand have demonstrated the ability to detect pyrogenicity in samples thathave passed the rabbit pyrogen test and the Limulus amebocyte lysatetest (Taktak et al, J. Pharm. Pharmacol. (1990), 43:578-82). In anadditional embodiment, the auris-acceptable otic therapeutic agentformulation is subject to depyrogenation. In a further embodiment, theprocess for the manufacture of the auris-acceptable otic therapeuticagent formulation comprises testing the formulation for pyrogenicity. Incertain embodiments, the formulations described herein are substantiallyfree of pyrogens.

pH and Practical Osmolarity

As used herein, “practical osmolarity” means the osmolarity of aformulation that is measured by including the active agent and allexcipients except the gelling and/or the thickening agent (e.g.,polyoxyethylene-polyooxypropylene copolymers, carboxymethylcellulose orthe like). The practical osmolarity of a formulation described herein ismeasured by any suitable method, e.g., a freezing point depressionmethod as described in Viegas et. al., Int. J. Pharm., 1998, 160,157-162. In some instances, the practical osmolarity of a compositiondescribed herein is measured by vapor pressure osmometry (e.g., vaporpressure depression method) that allows for determination of theosmolarity of a composition at higher temperatures. In some instances,vapor pressure depression method allows for determination of theosmolarity of a formulation comprising a gelling agent (e.g., athermoreversible polymer) at a higher temperature wherein the gellingagent is in the form of a gel. The practical osmolality of an oticformulation described herein is from about 100 mOsm/kg to about 1000mOsm/kg, from about 200 mOsm/kg to about 800 mOsm/kg, from about 250mOsm/kg to about 500 mOsm/kg, or from about 250 mOsm/kg to about 320mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about280 mOsm/kg to about 320 mOsm/kg. In some embodiments, the formulationsdescribed herein have a practical osmolarity of about 100 mOsm/L toabout 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about250 mOsm/L to about 320 mOsm/L, or about 280 mOsm/L to about 320 mOsm/L.

In some embodiments, the osmolarity at a target site of action (e.g.,the perilymph) is about the same as the delivered osmolarity (i.e.,osmolarity of materials that cross or penetrate the round windowmembrane) of any formulation described herein. In some embodiments, theformulations described herein have a deliverable osmolarity of about 150mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 500 mOsm/L, about250 mOsm/L to about 350 mOsm/L, about 280 mOsm/L to about 370 mOsm/L orabout 250 mOsm/L to about 320 mOsm/L.

The main cation present in the endolymph is potassium. In addition theendolymph has a high concentration of positively charged amino acids.The main cation present in the perilymph is sodium. In certaininstances, the ionic composition of the endolymph and perilymph regulatethe electrochemical impulses of hair cells. In certain instances, anychange in the ionic balance of the endolymph or perilymph results in aloss of hearing due to changes in the conduction of electrochemicalimpulses along otic hair cells. In some embodiments, a compositiondisclosed herein does not disrupt the ionic balance of the perilymph. Insome embodiments, a composition disclosed herein has an ionic balancethat is the same as or substantially the same as the perilymph. In someembodiments, a composition disclosed herein does not disrupt the ionicbalance of the endolymph. In some embodiments, a composition disclosedherein has an ionic balance that is the same as or substantially thesame as the endolymph. In some embodiments, an otic formulationdescribed herein is formulated to provide an ionic balance that iscompatible with inner ear fluids (e.g., endolymph and/or perilymph).

The endolymph and the perilymph have a pH that is close to thephysiological pH of blood. The endolymph has a pH range of about7.2-7.9; the perilymph has a pH range of about 7.2-7.4. The in situ pHof the proximal endolymph is about 7.4 while the pH of distal endolymphis about 7.9.

In some embodiments, the pH of a composition described herein isadjusted (e.g., by use of a buffer) to an endolymph-compatible pH rangeof about 5.5 to 9.0. In specific embodiments, the pH of a compositiondescribed herein is adjusted to a perilymph-suitable pH range of about5.5 to about 9.0. In some embodiments, the pH of a composition describedherein is adjusted to a perilymph-suitable range of about 5.5 to about8.0, about 6 to about 8.0 or about 6.6 to about 8.0. In someembodiments, the pH of a composition described herein is adjusted to aperilymph-suitable pH range of about 7.0-7.6.

In some embodiments, useful formulations also include one or more pHadjusting agents or buffering agents. Suitable pH adjusting agents orbuffers include, but are not limited to acetate, bicarbonate, ammoniumchloride, citrate, phosphate, pharmaceutically acceptable salts thereofand combinations or mixtures thereof.

In one embodiment, when one or more buffers are utilized in theformulations of the present disclosure, they are combined, e.g., with apharmaceutically acceptable vehicle and are present in the finalformulation, e.g., in an amount ranging from about 0.1% to about 20%,from about 0.5% to about 10%. In certain embodiments of the presentdisclosure, the amount of buffer included in the gel formulations are anamount such that the pH of the gel formulation does not interfere withthe body's natural buffering system.

In one embodiment, diluents are also used to stabilize compounds becausethey can provide a more stable environment. Salts dissolved in bufferedsolutions (which also can provide pH control or maintenance) areutilized as diluents in the art, including, but not limited to aphosphate buffered saline solution.

In some embodiments, any gel formulation described herein has a pH thatallows for sterilization (e.g., by filtration or aseptic mixing or heattreatment and/or autoclaving (e.g., terminal sterilization) of a gelformulation without degradation of the pharmaceutical agent (e.g., aurissensory cell modulating agent) or the polymers comprising the gel. Inorder to reduce hydrolysis and/or degradation of the otic agent and/orthe gel polymer during sterilization, the buffer pH is designed tomaintain pH of the formulation in the 7-8 range during the process ofsterilization (e.g., high temperature autoclaving).

In specific embodiments, any gel formulation described herein has a pHthat allows for terminal sterilization (e.g., by heat treatment and/orautoclaving) of a gel formulation without degradation of thepharmaceutical agent (e.g., auris sensory cell modulating agent) or thepolymers comprising the gel. For example, in order to reduce hydrolysisand/or degradation of the otic agent and/or the gel polymer duringautoclaving, the buffer pH is designed to maintain pH of the formulationin the 7-8 range at elevated temperatures. Any appropriate buffer isused depending on the otic agent used in the formulation. In someinstances, since pK_(a) of TRIS decreases as temperature increases atapproximately −0.03/° C. and pK_(a) of PBS increases as temperatureincreases at approximately 0.003/° C., autoclaving at 250° F. (121° C.)results in a significant downward pH shift (i.e. more acidic) in theTRIS buffer whereas a relatively much less upward pH shift in the PBSbuffer and therefore much increased hydrolysis and/or degradation of anotic agent in TRIS than in PBS. Degradation of an otic agent is reducedby the use of an appropriate combination of a buffer and polymericadditives (e.g. CMC) as described herein.

In some embodiments, a formulation pH of between about 5.0 and about9.0, between about 5.5 and about 8.5, between about 6.0 and about 7.6,between about 7 and about 7.8, between about 7.0 and about 7.6, betweenabout 7.2 and 7.6, or between about 7.2 and about 7.4 is suitable forsterilization (e.g., by filtration or aseptic mixing or heat treatmentand/or autoclaving (e.g., terminal sterilization)) of auris formulationsdescribed herein. In specific embodiments a formulation pH of about 6.0,about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about7.5, or about 7.6 is suitable for sterilization (e.g., by filtration oraseptic mixing or heat treatment and/or autoclaving (e.g., terminalsterilization)) of any composition described herein.

In some embodiments, the formulations have a pH as described herein, andinclude a thickening agent (e.g., a viscosity enhancing agent) such as,by way of non-limiting example, a cellulose based thickening agentdescribed herein. In some instances, the addition of a secondary polymer(e.g., a thickening agent) and a pH of formulation as described herein,allows for sterilization of a formulation described herein without anysubstantial degradation of the otic agent and/or the polymer componentsin the otic formulation. In some embodiments, the ratio of athermoreversible poloxamer to a thickening agent in a formulation thathas a pH as described herein, is about 40:1, about 35:1, about 30:1,about 25:1, about 20:1, about 15:1 about 10:1, or about 5:1. Forexample, in certain embodiments, a sustained and/or extended releaseformulation described herein comprises a combination of poloxamer 407(pluronic F127) and carboxymethylcellulose (CMC) in a ratio of about40:1, about 35:1, about 30:1, about 25:1, about 20:1, about 15:1, about10:1 or about 5:1.

In some embodiments, the amount of thermoreversible polymer in anyformulation described herein is about 10%, about 15%, about 20%, about25%, about 30%, about 35% or about 40% of the total weight of theformulation. In some embodiments, the amount of thermoreversible polymerin any formulation described herein is about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24% or about 25%of the total weight of the formulation. In some embodiments, the amountof thermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 7.5% of the total weight of the formulation.In some embodiments, the amount of thermoreversible polymer (e.g.,pluronic F127) in any formulation described herein is about 10% of thetotal weight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 11% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 12% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 13% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 14% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 15% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 16% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 17% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 18% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 19% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 20% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 21% of the total weight of the formulation. Insome embodiments, the amount of thermoreversible polymer (e.g., pluronicF127) in any formulation described herein is about 23% of the totalweight of the formulation. In some embodiments, the amount ofthermoreversible polymer (e.g., pluronic F127) in any formulationdescribed herein is about 25% of the total weight of the formulation.

In some embodiments, the amount of thickening agent (e.g., a gellingagent) in any formulation described herein is about 1%, about 5%, about10%, or about 15% of the total weight of the formulation. In someembodiments, the amount of thickening agent (e.g., a gelling agent) inany formulation described herein is about 0.5%, about 1%, about 1.5%,about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, orabout 5% of the total weight of the formulation.

In some embodiments, the pharmaceutical formulations described hereinare stable with respect to pH over a period of any of at least about 1day, at least about 2 days, at least about 3 days, at least about 4days, at least about 5 days, at least about 6 days, at least about 1week, at least about 2 weeks, at least about 3 weeks, at least about 4weeks, at least about 5 weeks, at least about 6 weeks, at least about 7weeks, at least about 8 weeks, at least about 1 month, at least about 2months, at least about 3 months, at least about 4 months, at least about5 months, or at least about 6 months. In other embodiments, theformulations described herein are stable with respect to pH over aperiod of at least about 1 week. Also described herein are formulationsthat are stable with respect to pH over a period of at least about 1month.

Tonicity Agents

In general, the endolymph has a higher osmolality than the perilymph.For example, the endolymph has an osmolality of about 304 mOsm/kg H₂Owhile the perilymph has an osmolality of about 294 mOsm/kg H₂O. Incertain embodiments, tonicity agents are added to the formulationsdescribed herein in an amount as to provide a practical osmolality of anotic formulation of about 100 mOsm/kg to about 1000 mOsm/kg, from about200 mOsm/kg to about 800 mOsm/kg, from about 250 mOsm/kg to about 500mOsm/kg, or from about 250 mOsm/kg to about 350 mOsm/kg or from about280 mOsm/kg to about 320 mOsm/kg. In some embodiments, the formulationsdescribed herein have a practical osmolarity of about 100 mOsm/L toabout 1000 mOsm/L, about 200 mOsm/L to about 800 mOsm/L, about 250mOsm/L to about 500 mOsm/L, about 250 mOsm/L to about 350 mOsm/L, about280 mOsm/L to about 320 mOsm/L or about 250 mOsm/L to about 320 mOsm/L.

In some embodiments, the deliverable osmolarity of any formulationdescribed herein is designed to be isotonic with the targeted oticstructure (e.g., endolymph, perilymph or the like). In specificembodiments, auris compositions described herein are formulated toprovide a delivered perilymph-suitable osmolarity at the target site ofaction of about 250 to about 320 mOsm/L; and preferably about 270 toabout 320 mOsm/L. In specific embodiments, auris compositions describedherein are formulated to provide a delivered perilymph-suitableosmolality at the target site of action of about 250 to about 320mOsm/kg H₂O; or an osmolality of about 270 to about 320 mOsm/kg H₂O. Inspecific embodiments, the deliverable osmolarity/osmolality of theformulations (i.e., the osmolarity/osmolality of the formulation in theabsence of gelling or thickening agents (e.g., thermoreversible gelpolymers) is adjusted, for example, by the use of appropriate saltconcentrations (e.g., concentration of potassium or sodium salts) or theuse of tonicity agents which renders the formulationsendolymph-compatible and/or perilymph-compatible (i.e. isotonic with theendolymph and/or perilymph) upon delivery at the target site. Theosmolarity of a formulation comprising a thermoreversible gel polymer isan unreliable measure due to the association of varying amounts of waterwith the monomeric units of the polymer. The practical osmolarity of aformulation (i.e., osmolarity in the absence of a gelling or thickeningagent (e.g. a thermoreversible gel polymer) is a reliable measure and ismeasured by any suitable method (e.g., freezing point depression method,vapor depression method). In some instances, the formulations describedherein provide a deliverable osmolarity (e.g., at a target site (e.g.,perilymph) that causes minimal disturbance to the environment of theinner ear and causes minimum discomfort (e.g., vertigo and/or nausea) toa mammal upon administration.

In some embodiments, any formulation described herein is isotonic withthe perilymph and/or endolymph. Isotonic formulations are provided bythe addition of a tonicity agent. Suitable tonicity agents include, butare not limited to any pharmaceutically acceptable sugar, salt or anycombinations or mixtures thereof, such as, but not limited to dextrose,glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.

Useful auris compositions include one or more salts in an amountrequired to bring osmolality of the composition into an acceptablerange. Such salts include those having sodium, potassium or ammoniumcations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some embodiments, the formulations described herein have a pH and/orpractical osmolarity as described herein, and have a concentration ofactive pharmaceutical ingredient between about 1 μM and about 10 μM,between about 1 mM and about 100 mM, between about 0.1 mM and about 100mM, between about 0.1 mM and about 100 nM. In some embodiments, theformulations described herein have a pH and/or practical osmolarity asdescribed herein, and have a concentration of active pharmaceuticalingredient between about 0.01%-about 20%, between about 0.01%-about10%., between about 0.01%-about 7.5%, between about 0.01%-6%, betweenabout 0.01-5%, between about 0.1-about 10%, or between about 0.1-about6% of the active ingredient by weight of the formulation. In someembodiments, the formulations described herein have a pH and/orpractical osmolarity as described herein, and have a concentration ofactive pharmaceutical ingredient between about 0.1 and about 70 mg,between about 1 mg and about 70 mg/mL, between about 1 mg and about 50mg/mL, between about 1 mg/mL and about 20 mg/mL, between about 1 mg/mLto about 10 mg/mL, between about 1 mg/mL to about 5 mg/mL, or betweenabout 0.5 mg/mL to about 5 mg/mL of the active agent by volume of theformulation. In some embodiments, the formulations described herein havea pH and/or practical osmolarity as described herein, and have aconcentration of active pharmaceutical ingredient between about 1 μg/mLand about 500 μg/mL, between about 1 μg/mL and about 250 μg/mL, betweenabout 1 μg and about 100 μg/mL, between about 1 μg/mL and about 50μg/mL, or between about 1 μg/mL and about 20 μg/mL of the active agentby volume of the formulation.

Particle Size

Size reduction is used to increase surface area and/or modulateformulation dissolution properties. It is also used to maintain aconsistent average particle size distribution (PSD) (e.g.,micrometer-sized particles, nanometer-sized particles or the like) forany formulation described herein. In some embodiments, any formulationdescribed herein comprises multiparticulates, i.e., a plurality ofparticle sizes (e.g., micronized particles, nano-sized particles,non-sized particles, colloidal particles); i.e., the formulation is amultiparticulate formulation. In some embodiments, any formulationdescribed herein comprises one or more multiparticulate (e.g.,micronized) therapeutic agents. Micronization is a process of reducingthe average diameter of particles of a solid material. Micronizedparticles are from about micrometer-sized in diameter to aboutnanometer-sized in diameter. In some embodiments, the average diameterof particles in a micronized solid is from about 0.5 μm to about 500 μm.In some embodiments, the average diameter of particles in a micronizedsolid is from about 1 μm to about 200 μm. In some embodiments, theaverage diameter of particles in a micronized solid is from about 2 μmto about 100 μm. In some embodiments, the average diameter of particlesin a micronized solid is from about 3 μm to about 50 μm. In someembodiments, a particulate micronized solid comprises particle sizes ofless than about 5 microns, less than about 20 microns and/or less thanabout 100 microns. In some embodiments, the use of particulates (e.g.,micronized particles) of auris sensory cell modulating agent allows forextended and/or sustained release of the auris sensory cell modulatingagent from any formulation described herein compared to a formulationcomprising non-multiparticulate (e.g., non-micronized) auris sensorycell modulating agent. In some instances, formulations containingmultiparticulate (e.g. micronized) auris sensory cell modulating agentare ejected from a 1 mL syringe adapted with a 27G needle without anyplugging or clogging.

In some instances, any particle in any formulation described herein is acoated particle (e.g., a coated micronized particle, nano-particle)and/or a microsphere and/or a liposomal particle. Particle sizereduction techniques include, by way of example, grinding, milling(e.g., air-attrition milling jet milling), ball milling), coacervation,complex coacervation, high pressure homogenization, spray drying and/orsupercritical fluid crystallization. In some instances, particles aresized by mechanical impact (e.g., by hammer mills, ball mill and/or pinmills). In some instances, particles are sized via fluid energy (e.g.,by spiral jet mills, loop jet mills, and/or fluidized bed jet mills). Insome embodiments formulations described herein comprise crystallineparticles and/or isotropic particles. In some embodiments, formulationsdescribed herein comprise amorphous particles and/or anisotropicparticles. In some embodiments, formulations described herein comprisetherapeutic agent particles wherein the therapeutic agent is a freebase, or a salt, or a prodrug of a therapeutic agent, or any combinationthereof.

In some embodiments, a formulation described herein comprises one ormore auris sensory cell modulating agents wherein the auris sensory cellmodulating agent comprises nanoparticulates. In some embodiments, aformulation described herein comprises auris sensory cell modulatingagent beads (e.g., dextromethorphan beads) that are optionally coatedwith controlled release excipients. In some embodiments, a formulationdescribed herein comprises an auris sensory cell modulating agent thatis granulated and/or reduced in size and coated with controlled releaseexcipients; the granulated coated auris sensory cell modulating agentparticulates are then optionally micronized and/or formulated in any ofthe compositions described herein.

In some instances, a combination of an auris sensory cell modulatingagent as a neutral molecule, free acid or free base and a salt of theauris sensory cell modulating agent is used to prepare pulsed releaseotic agent formulations using the procedures described herein. In someformulations, a combination of a micronized auris sensory cellmodulating agent (and/or salt or prodrug thereof) and coated particles(e.g., nanoparticles, liposomes, microspheres) is used to prepare pulsedrelease otic agent formulations using any procedure described herein.Alternatively, a pulsed release profile is achieved by solubilizing upto 20% of the delivered dose of the auris sensory cell modulating agent(e.g., micronized auris sensory cell modulating agent, free base, freeacid or salt or prodrug thereof; multiparticulate auris sensory cellmodulating agent, free base, free acid or salt or prodrug thereof) withthe aid of cyclodextrins, surfactants (e.g., poloxamers (407, 338, 188),tween (80, 60, 20, 81), PEG-hydrogenated castor oil, cosolvents likeN-methyl-2-Pyrrolidone or the like and preparing pulsed releaseformulations using any procedure described herein.

In specific embodiments, any auris-compatible formulation describedherein comprises one or more micronized pharmaceutical agents (e.g.,auris sensory cell modulating agents). In some of such embodiments, amicronized pharmaceutical agent comprises micronized particles, coated(e.g., with an extended release coat) micronized particles, or acombination thereof. In some of such embodiments, a micronizedpharmaceutical agent comprising micronized particles, coated micronizedparticles, or a combination thereof, comprises an auris sensory cellmodulating agent as a neutral molecule, a free acid, a free base, asalt, a prodrug or any combination thereof. In certain embodiments, apharmaceutical composition described herein comprises an auris sensorycell modulating agent as a micronized powder. In certain embodiments, apharmaceutical composition described herein comprises an auris sensorycell modulating agent in the form of a micro-auris sensory cellmodulating agent powder.

The multiparticulates and/or micronized auris sensory cell modulatingagents described herein are delivered to an auris structure (e.g., innerear) by means of any type of matrix including solid, liquid or gelmatrices. In some embodiments, the multiparticulates and/or micronizedauris sensory cell modulating agents described herein are delivered toan auris structure (e.g., inner ear) by means of any type of matrixincluding solid, liquid or gel matrices via intratympanic injection.

Tunable Release Characteristics

The release of active agent from any formulation, composition or devicedescribed herein is optionally tunable to the desired releasecharacteristics. In some embodiments, a composition described herein isa solution that is substantially free of gelling components. In suchinstances, the composition provides essentially immediate release of anactive agent. In some of such embodiments, the composition is useful inperfusion of otic structures, e.g., during surgery.

In some embodiments, a composition described herein is a solution thatis substantially free of gelling components and comprises micronizedotic agent (e.g., a corticosteroid). In some of such embodiments, thecomposition provides release of an active agent from about 2 days toabout 4 days. In some embodiments, a composition described hereincomprises a gelling agent (e.g., poloxamer 407) and provides release ofan active agent over a period of from about 1 day to about 3 days. Insome embodiments, a composition described herein comprises a gellingagent (e.g., poloxamer 407) and provides release of an active agent overa period of from about 1 day to about 5 days. In some embodiments, acomposition described herein comprises a gelling agent (e.g., poloxamer407) and provides release of an active agent over a period of from about2 days to about 7 days.

In some embodiments, a composition described herein comprises a gellingagent (e.g., poloxamer 407) in combination with micronized otic agentand provides extended sustained release over a longer period of time. Insome embodiments, a composition described herein comprises about 14-17%of a gelling agent (e.g., poloxamer 407) and micronized otic agent, andprovides extended sustained release over a period of from about 1 weekto about 3 weeks. In some embodiments, a composition described hereincomprises about 18-21% of a gelling agent (e.g., poloxamer 407) andmicronized otic agent, and provides extended sustained release over aperiod of from about 3 weeks to about 6 weeks.

Accordingly, the amount of gelling agent in a composition, and theparticle size of an otic agent are tunable to the desired releaseprofile of an otic agent from the composition.

As described herein, compositions comprising micronized otic agentsprovide extended release over a longer period of time compared tocompositions comprising non-micronized otic agents. In some instances,the micronized otic agent provides a steady supply (e.g., +/−20%) ofactive agent via slow degradation and serves as a depot for the activeagent; such a depot effect increases residence time of the otic agent inthe ear. In specific embodiments, selection of an appropriate particlesize of the active agent (e.g., micronized active agent) in combinationwith the amount of gelling agent in the composition provides tunableextended release characteristics that allow for release of an activeagent over a period of hours, days, weeks or months.

In some embodiments, the viscosity of any formulation described hereinis designed to provide a suitable rate of release from an auriscompatible gel. In some embodiments, the concentration of a thickeningagent (e.g., gelling components such as polyoxyethylene-polyoxypropylenecopolymers) allows for a tunable mean dissolution time (MDT). The MDT isinversely proportional to the release rate of an active agent from acomposition or device described herein. Experimentally, the releasedotic agent is optionally fitted to the Korsmeyer-Peppas equation

$\frac{Q}{Q_{\alpha}} = {{kt}^{n} + b}$

where Q is the amount of otic agent released at time t, Q_(α) is theoverall released amount of otic agent, k is a release constant of thenth order, n is a dimensionless number related to the dissolutionmechanism and b is the axis intercept, characterizing the initial burstrelease mechanism wherein n=1 characterizes an erosion controlledmechanism. The mean dissolution time (MDT) is the sum of differentperiods of time the drug molecules stay in the matrix before release,divided by the total number of molecules and is optionally calculatedby:

${MDT} = \frac{{nk}^{{- 1}/n}}{n + 1}$

For example, a linear relationship between the mean dissolution time(MDT) of a composition or device and the concentration of the gellingagent (e.g., poloxamer) indicates that the otic agent is released due tothe erosion of the polymer gel (e.g., poloxamer) and not via diffusion.In another example, a non-linear relationship indicates release of oticagent via a combination of diffusion and/or polymer gel degradation. Inanother example, a faster gel elimination time course of a compositionor device (a faster release of active agent) indicates lower meandissolution time (MDT). The concentration of gelling components and/oractive agent in a composition are tested to determine suitableparameters for MDT. In some embodiments, injection volumes are alsotested to determine suitable parameters for preclinical and clinicalstudies. The gel strength and concentration of the active agent affectsrelease kinetics of an otic agent from the composition. At low poloxamerconcentration, elimination rate is accelerated (MDT is lower). Anincrease in otic agent concentration in the composition or deviceprolongs residence time and/or MDT of the otic agent in the ear.

In some embodiments, the MDT for poloxamer from a composition or devicedescribed herein is at least 6 hours. In some embodiments, the MDT forpoloxamer from a composition or device described herein is at least 10hours.

In some embodiments, the MDT for an active agent from a composition ordevice described herein is from about 30 hours to about 48 hours. Insome embodiments, the MDT for an active agent from a composition ordevice described herein is from about 30 hours to about 96 hours. Insome embodiments, the MDT for an active agent from a composition ordevice described herein is from about 30 hours to about 1 week. In someembodiments, the MDT for an active agent from a composition or devicedescribed herein is from about 1 week to about 6 weeks.

In certain embodiments, any controlled release otic formulationdescribed herein increases the exposure of an otic agent and increasesthe Area Under the Curve (AUC) in otic fluids (e.g., endolymph and/orperilymph) by about 30%, about 40%, about 50%, about 60%, about 70%,about 80% or about 90% compared to a formulation that is not acontrolled release otic formulation. In certain embodiments, anycontrolled release otic formulation described herein increases theexposure time of an otic agent and decreases the C_(max) in otic fluids(e.g., endolymph and/or perilymph) by about 40%, about 30%, about 20%,or about 10%, compared to a formulation that is not a controlled releaseotic formulation. In certain embodiments, any controlled release oticformulation described herein alters (e.g. reduces) the ratio of C_(max)to C_(min) compared to a formulation that is not a controlled releaseotic formulation. In certain embodiments, any controlled release oticformulation described herein increases the exposure of an otic agent andincreases the length of time that the concentration of an otic agent isabove C_(min) by about 30%, about 40%, about 50%, about 60%, about 70%,about 80% or about 90% compared to a formulation that is not acontrolled release otic formulation. In certain instances, controlledrelease formulations described herein delay the time to C_(max). Incertain instances, the controlled steady release of a drug prolongs thetime the concentration of the drug will stay above the C_(min). In someembodiments, auris compositions described herein prolong the residencetime of a drug in the inner ear and provide a stable drug exposureprofile. In some instances, an increase in concentration of an activeagent in the composition saturates the clearance process and allows fora more rapid and stable steady state to be reached.

In certain instances, once drug exposure (e.g., concentration in theendolymph or perilymph) of a drug reaches steady state, theconcentration of the drug in the endolymph or perilymph stays at orabout the therapeutic dose for an extended period of time (e.g., oneday, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week, 3 weeks, 6weeks, 2 months). In some embodiments, the steady state concentration ofactive agent released from a controlled release formulation describedherein is about 20 to about 50 times the steady state concentration ofan active agent released from a formulation that is not a controlledrelease formulation. FIG. 5 shows predicted tunable release of an activeagent from four compositions

Pharmaceutical Formulations

Provided herein are pharmaceutical compositions or devices that includeat least one auris sensory cell modulating agent and a pharmaceuticallyacceptable diluent(s), excipient(s), or carrier(s). In some embodiments,the pharmaceutical compositions include other medicinal orpharmaceutical agents, carriers, adjuvants, such as preserving,stabilizing, wetting or emulsifying agents, solution promoters, saltsfor regulating the osmotic pressure, and/or buffers. In otherembodiments, the pharmaceutical compositions also contain othertherapeutic substances.

In some embodiments, the compositions or devices described hereininclude a dye to help enhance the visualization of the gel when applied.In some embodiments, dyes that are compatible with the auris-acceptablecompositions or devices described herein include Evans blue (e.g., 0.5%of the total weight of an otic formulation), Methylene blue (e.g., 1% ofthe total weight of an otic formulation), Isosulfan blue (e.g., 1% ofthe total weight of an otic formulation), Trypan blue (e.g., 0.15% ofthe total weight of an otic formulation), and/or indocyanine green(e.g., 25 mg/vial). Other common dyes, e.g., FD&C red 40, FD&C red 3,FD&C yellow 5, FD&C yellow 6, FD&C blue 1, FD&C blue2, FD&C green 3,fluorescence dyes (e.g., Fluorescein isothiocyanate, rhodamine, AlexaFluors, DyLight Fluors) and/or dyes that are visualizable in conjunctionwith non-invasive imaging techniques such as MRI, CAT scans, PET scansor the like. Gadolinium-based MRI dyes, iodine-base dyes, barium-baseddyes or the like are also contemplated for use with any otic formulationdescribed herein. Other dyes that are compatible with any formulation orcomposition described herein are listed in the Sigma-Aldrich catalogunder dyes (which is included herein by reference for such disclosure).

In some embodiments, mechanical or imaging devices are used to monitoror survey the hearing, balance or other auris disorder. For example,magnetic resonance imaging (MRI) devices are specifically contemplatedwithin the scope of the embodiments, wherein the MRI devices (forexample, 3 Tesla MRI devices) are capable of evaluating Meniere Diseaseprogression, and subsequent treatment with the pharmaceuticalformulations disclosed herein. Gadolinium-based dyes, iodine-base dyes,barium-based dyes or the like are also contemplated for use with anyauris-compatible composition or device described herein and/or with anymechanical or imaging devices described herein. In certain embodiments,gadolinium hydrate is used in combination with MRI and/or anypharmaceutical composition or device described herein to evaluatedisease severity (e.g., size of endolymphatic hydrops), formulationpenetration into the inner ear, and/or therapeutic effectiveness of thepharmaceutical formulations/devices in the otic diseases describedherein (e.g., Meniere's disease).

Any pharmaceutical composition or device described herein isadministered by locating the composition or device in contact with thecrista fenestrae cochlea, the round window, the tympanic cavity, thetympanic membrane, the auris media or the auris externa.

In one specific embodiment of the auris-acceptable controlled releaseauris sensory cell modulating agent pharmaceutical formulationsdescribed herein, the auris sensory cell modulating agent is provided ina gel matrix, also referred to herein as “auris acceptable gelformulations,” “auris interna-acceptable gel formulations,” “aurismedia-acceptable gel formulations,” “auris externa-acceptable gelformulations”, “auris gel formulations” or variations thereof. All ofthe components of the gel formulation must be compatible with thetargeted auris structure. Further, the gel formulations providecontrolled release of the auris sensory cell modulating agent to thedesired site within the targeted auris structure; in some embodiments,the gel formulation also has an immediate or rapid release component fordelivery of the auris sensory cell modulating agent to the desiredtarget site. In other embodiments, the gel formulation has a sustainedrelease component for delivery of the auris sensory cell modulatingagent. In some embodiments, the gel formulation comprises amultiparticulate (e.g., micronized) auris sensory cell modulating agent.In some embodiments, the auris gel formulations are biodegradable. Inother embodiments, the auris gel formulations include a mucoadhesiveexcipient to allow adhesion to the external mucous layer of the roundwindow membrane. In yet other embodiments, the auris gel formulationsinclude a penetration enhancer excipient; in further embodiments, theauris gel formulation contains a viscosity enhancing agent sufficient toprovide a viscosity of between about 500 and 1,000,000 centipoise,between about 750 and 1,000,000 centipoise; between about 1000 and1,000,000 centipoise; between about 1000 and 400,000 centipoise; betweenabout 2000 and 100,000 centipoise; between about 3000 and 50,000centipoise; between about 4000 and 25,000 centipoise; between about 5000and 20,000 centipoise; or between about 6000 and 15,000 centipoise. Insome embodiments, the auris gel formulation contains a viscosityenhancing agent sufficient to provide a viscosity of between about50,0000 and 1,000,000 centipoise.

In some embodiments, the compositions or devices described herein arelow viscosity compositions or devices at body temperature. In someembodiments, low viscosity compositions or devices contain from about 1%to about 10% of a viscosity enhancing agent (e.g., gelling componentssuch as polyoxyethylene-polyoxypropylene copolymers). In someembodiments, low viscosity compositions or devices contain from about 2%to about 10% of a viscosity enhancing agent (e.g., gelling componentssuch as polyoxyethylene-polyoxypropylene copolymers). In someembodiments, low viscosity compositions or devices contain from about 5%to about 10% of a viscosity enhancing agent (e.g., gelling componentssuch as polyoxyethylene-polyoxypropylene copolymers). In someembodiments, low viscosity compositions or devices are substantiallyfree of a viscosity enhancing agent (e.g., gelling components such aspolyoxyethylene-polyoxypropylene copolymers). In some embodiments, a lowviscosity auris sensory cell modulator composition or device describedherein provides an apparent viscosity of from about 100 cP to about10,000 cP. In some embodiments, a low viscosity auris sensory cellmodulator composition or device described herein provides an apparentviscosity of from about 500 cP to about 10,000 cP. In some embodiments,a low viscosity auris sensory cell modulator composition or devicedescribed herein provides an apparent viscosity of from about 1000 cP toabout 10,000 cP. In some of such embodiments, a low viscosity aurissensory cell modulator composition or device is administered incombination with an external otic intervention, e.g., a surgicalprocedure including but not limited to middle ear surgery, inner earsurgery, typanostomy, cochleostomy, labyrinthotomy, mastoidectomy,stapedectomy, stapedotomy, endolymphatic sacculotomy or the like. Insome of such embodiments, a low viscosity auris sensory cell modulatorcomposition or device is administered during an otic intervention. Inother such embodiments, a low viscosity auris sensory cell modulatorcomposition or device is administered before the otic intervention.

In some embodiments, the compositions or devices described herein arehigh viscosity compositions or devices at body temperature. In someembodiments, high viscosity compositions or devices contain from about10% to about 25% of a viscosity enhancing agent (e.g., gellingcomponents such as polyoxyethylene-polyoxypropylene copolymers). In someembodiments, high viscosity compositions or devices contain from about14% to about 22% of a viscosity enhancing agent (e.g., gellingcomponents such as polyoxyethylene-polyoxypropylene copolymers). In someembodiments, high viscosity compositions or devices contain from about15% to about 21% of a viscosity enhancing agent (e.g., gellingcomponents such as polyoxyethylene-polyoxypropylene copolymers). In someembodiments, a high viscosity auris sensory cell modulator compositionor device described herein provides an apparent viscosity of from about100,000 cP to about 1,000,000 cP. In some embodiments, a high viscosityauris sensory cell modulator composition or device described hereinprovides an apparent viscosity of from about 150,000 cP to about 500,000cP. In some embodiments, a high viscosity auris sensory cell modulatorcomposition or device described herein provides an apparent viscosity offrom about 250,000 cP to about 500,000 cP. In some of such embodiments,a high viscosity composition or device is a liquid at room temperatureand gels at about between room temperature and body temperature(including an individual with a serious fever, e.g., up to about 42°C.). In some embodiments, an auris sensory cell modulator high viscositycomposition or device is administered as monotherapy for treatment of anotic disease or condition described herein. In some embodiments, anauris sensory cell modulator high viscosity composition or device isadministered in combination with an external otic intervention, e.g., asurgical procedure including but not limited to middle ear surgery,inner ear surgery, typanostomy, cochleostomy, labyrinthotomy,mastoidectomy, stapedectomy, stapedotomy, endolymphatic sacculotomy orthe like. In some of such embodiments, a high viscosity auris sensorycell modulator composition or device is administered after the oticintervention. In other such embodiments, a high viscosity auris sensorycell modulator composition or device is administered before the oticintervention.

In other embodiments, the auris interna pharmaceutical formulationsdescribed herein further provide an auris-acceptable hydrogel; in yetother embodiments, the auris pharmaceutical formulations provide anauris-acceptable microsphere or microparticle; in still otherembodiments, the auris pharmaceutical formulations provide anauris-acceptable liposome. In some embodiments, the auris pharmaceuticalformulations provide an auris-acceptable foam; in yet other embodiments,the auris pharmaceutical formulations provide an auris-acceptable paint;in still further embodiments, the auris pharmaceutical formulationsprovide an auris-acceptable in situ forming spongy material. In someembodiments, the auris pharmaceutical formulations provide anauris-acceptable solvent release gel. In some embodiments, the aurispharmaceutical formulations provide an actinic radiation curable gel.Further embodiments include a thermoreversible gel in the aurispharmaceutical formulation, such that upon preparation of the gel atroom temperature or below, the formulation is a fluid, but uponapplication of the gel into or near the auris interna and/or auris mediatarget site, including the tympanic cavity, round window membrane or thecrista fenestrae cochleae, the auris-pharmaceutical formulation stiffensor hardens into a gel-like substance.

In further or alternative embodiments, the auris gel formulations arecapable of being administered on or near the round window membrane viaintratympanic injection. In other embodiments, the auris gelformulations are administered on or near the round window or the cristafenestrae cochleae through entry via a post-auricular incision andsurgical manipulation into or near the round window or the cristafenestrae cochleae area. Alternatively, the auris gel formulation isapplied via syringe and needle, wherein the needle is inserted throughthe tympanic membrane and guided to the area of the round window orcrista fenestrae cochleae. The auris gel formulations are then depositedon or near the round window or crista fenestrae cochleae for localizedtreatment of autoimmune otic disorders. In other embodiments, the aurisgel formulations are applied via microcathethers implanted into thepatient, and in yet further embodiments the formulations areadministered via a pump device onto or near the round window membrane.In still further embodiments, the auris gel formulations are applied ator near the round window membrane via a microinjection device. In yetother embodiments, the auris gel formulations are applied in thetympanic cavity. In some embodiments, the auris gel formulations areapplied on the tympanic membrane. In still other embodiments, the aurisgel formulations are applied onto or in the auditory canal.

In further specific embodiments, any pharmaceutical composition ordevice described herein comprises a multiparticulate auris sensory cellmodulating agent in a liquid matrix (e.g., a liquid composition forintratympanic injection, or otic drops). In certain embodiments, anypharmaceutical composition described herein comprises a multiparticulateauris sensory cell modulating agent in a solid matrix.

Controlled Release Formulations

In general, controlled release drug formulations impart control over therelease of drug with respect to site of release and time of releasewithin the body. As discussed herein, controlled release refers toimmediate release, delayed release, sustained release, extended release,variable release, pulsatile release and bi-modal release. Manyadvantages are offered by controlled release. First, controlled releaseof a pharmaceutical agent allows less frequent dosing and thus minimizesrepeated treatment. Second, controlled release treatment results in moreefficient drug utilization and less of the compound remains as aresidue. Third, controlled release offers the possibility of localizeddrug delivery by placement of a delivery device or formulation at thesite of disease. Still further, controlled release offers theopportunity to administer and release two or more different drugs, eachhaving a unique release profile, or to release the same drug atdifferent rates or for different durations, by means of a single dosageunit.

Accordingly, one aspect of the embodiments disclosed herein is toprovide a controlled release auris sensory cell modulating agentauris-acceptable composition or device for the treatment of autoimmunedisorders and/or inflammatory disorders. The controlled release aspectof the compositions and/or formulations and/or devices disclosed hereinis imparted through a variety of agents, including but not limited toexcipients, agents or materials that are acceptable for use in the aurisinterna or other otic structure. By way of example only, suchexcipients, agents or materials include an auris-acceptable polymer, anauris-acceptable viscosity enhancing agent, an auris-acceptable gel, anauris-acceptable paint, an auris-acceptable foam, an auris-acceptablexerogel, an auris-acceptable microsphere or microparticle, anauris-acceptable hydrogel, an auris-acceptable in situ forming spongymaterial, an auris-acceptable actinic radiation curable gel, anauris-acceptable solvent release gel, an auris-acceptable liposome, anauris-acceptable nanocapsule or nanosphere, an auris-acceptablethermoreversible gel, or combinations thereof.

Auris-Acceptable Gels

Gels, sometimes referred to as jellies, have been defined in variousways. For example, the United States Pharmacopoeia defines gels assemisolid systems consisting of either suspensions made up of smallinorganic particles or large organic molecules interpenetrated by aliquid. Gels include a single-phase or a two-phase system. Asingle-phase gel consists of organic macromolecules distributeduniformly throughout a liquid in such a manner that no apparentboundaries exist between the dispersed macromolecules and the liquid.Some single-phase gels are prepared from synthetic macromolecules (e.g.,carbomer) or from natural gums, (e.g., tragacanth). In some embodiments,single-phase gels are generally aqueous, but will also be made usingalcohols and oils. Two-phase gels consist of a network of small discreteparticles.

Gels can also be classified as being hydrophobic or hydrophilic. Incertain embodiments, the base of a hydrophobic gel consists of a liquidparaffin with polyethylene or fatty oils gelled with colloidal silica,or aluminum or zinc soaps. In contrast, the base of hydrophobic gelsusually consists of water, glycerol, or propylene glycol gelled with asuitable gelling agent (e.g., tragacanth, starch, cellulose derivatives,carboxyvinylpolymers, and magnesium-aluminum silicates). In certainembodiments, the rheology of the compositions or devices disclosedherein is pseudo plastic, plastic, thixotropic, or dilatant.

In one embodiment the enhanced viscosity auris-acceptable formulationdescribed herein is not a liquid at room temperature. In certainembodiments, the enhanced viscosity formulation is characterized by aphase transition between room temperature and body temperature(including an individual with a serious fever, e.g., up to about 42°C.). In some embodiments, the phase transition occurs at 1° C. belowbody temperature, at 2° C. below body temperature, at 3° C. below bodytemperature, at 4° C. below body temperature, at 6° C. below bodytemperature, at 8° C. below body temperature, or at 10° C. below bodytemperature. In some embodiments, the phase transition occurs at about15° C. below body temperature, at about 20° C. below body temperature orat about 25° C. below body temperature. In specific embodiments, thegelation temperature (Tgel) of a formulation described herein is about20° C., about 25° C., or about 30° C. In certain embodiments, thegelation temperature (Tgel) of a formulation described herein is about35° C., or about 40° C. In one embodiment, administration of anyformulation described herein at about body temperature reduces orinhibits vertigo associated with intratympanic administration of oticformulations. Included within the definition of body temperature is thebody temperature of a healthy individual, or an unhealthy individual,including an individual with a fever (up to ˜42° C.). In someembodiments, the pharmaceutical compositions or devices described hereinare liquids at about room temperature and are administered at or aboutroom temperature, reducing or ameliorating side effects such as, forexample, vertigo.

Polymers composed of polyoxypropylene and polyoxyethylene formthermoreversible gels when incorporated into aqueous solutions. Thesepolymers have the ability to change from the liquid state to the gelstate at temperatures close to body temperature, therefore allowinguseful formulations that are applied to the targeted auris structure(s).The liquid state-to-gel state phase transition is dependent on thepolymer concentration and the ingredients in the solution.

Poloxamer 407 (PF-127) is a nonionic surfactant composed ofpolyoxyethylene-polyoxypropylene copolymers. Other poloxamers include188 (F-68 grade), 237 (F-87 grade), 338 (F-108 grade). Aqueous solutionsof poloxamers are stable in the presence of acids, alkalis, and metalions. PF-127 is a commercially availablepolyoxyethylene-polyoxypropylene triblock copolymer of general formulaE106 P70 E106, with an average molar mass of 13,000. The polymer can befurther purified by suitable methods that will enhance gelationproperties of the polymer. It contains approximately 70% ethylene oxide,which accounts for its hydrophilicity. It is one of the series ofpoloxamer ABA block copolymers, whose members share the chemical formulashown below.

PF-127 is of particular interest since concentrated solutions (>20% w/w)of the copolymer are transformed from low viscosity transparentsolutions to solid gels on heating to body temperature. This phenomenon,therefore, suggests that when placed in contact with the body, the gelpreparation will form a semi-solid structure and a sustained releasedepot. Furthermore, PF-127 has good solubilizing capacity, low toxicityand is, therefore, considered a good medium for drug delivery systems.

In an alternative embodiment, the thermogel is a PEG-PLGA-PEG triblockcopolymer (Jeong et al, Nature (1997), 388:860-2; Jeong et al, J.Control. Release (2000), 63:155-63; Jeong et al, Adv. Drug Delivery Rev.(2002), 54:37-51). The polymer exhibits sol-gel behavior over aconcentration of about 5% w/w to about 40% w/w. Depending on theproperties desired, the lactide/glycolide molar ratio in the PLGAcopolymer ranges from about 1:1 to about 20:1. The resulting copolymersare soluble in water and form a free-flowing liquid at room temperature,but form a hydrogel at body temperature. A commercially availablePEG-PLGA-PEG triblock copolymer is RESOMER RGP t50106 manufactured byBoehringer Ingelheim. This material is composed of a PGLA copolymer of50:50 poly(DL-lactide-co-glycolide) and is 10% w/w of PEG and has amolecular weight of about 6000.

ReGel® is a tradename of MacroMed Incorporated for a class of lowmolecular weight, biodegradable block copolymers having reverse thermalgelation properties as described in U.S. Pat. Nos. 6,004,573, 6,117949,6,201,072, and 6,287,588. It also includes biodegradable polymeric drugcarriers disclosed in pending U.S. patent application Ser. Nos.09/906,041, 09/559,799 and 10/919,603. The biodegradable drug carriercomprises ABA-type or BAB-type triblock copolymers or mixtures thereof,wherein the A-blocks are relatively hydrophobic and comprisebiodegradable polyesters or poly(orthoester)_(s), and the B-blocks arerelatively hydrophilic and comprise polyethylene glycol (PEG), saidcopolymers having a hydrophobic content of between 50.1 to 83% by weightand a hydrophilic content of between 17 to 49.9% by weight, and anoverall block copolymer molecular weight of between 2000 and 8000Daltons. The drug carriers exhibit water solubility at temperaturesbelow normal mammalian body temperatures and undergo reversible thermalgelation to then exist as a gel at temperatures equal to physiologicalmammalian body temperatures. The biodegradable, hydrophobic A polymerblock comprises a polyester or poly(ortho ester), in which the polyesteris synthesized from monomers selected from the group consisting ofD,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid,L-lactic acid, glycolide, glycolic acid, ε-caprolactone,ε-hydroxyhexanoic acid, γ-butyrolactone, γ-hydroxybutyric acid,δ-valerolactone, δ-hydroxyvaleric acid, hydroxybutyric acids, malicacid, and copolymers thereof and having an average molecular weight ofbetween about 600 and 3000 Daltons. The hydrophilic B-block segment ispreferably polyethylene glycol (PEG) having an average molecular weightof between about 500 and 2200 Daltons.

Additional biodegradable thermoplastic polyesters include AtriGel®(provided by Atrix Laboratories, Inc.) and/or those disclosed, e.g., inU.S. Pat. Nos. 5,324,519; 4,938,763; 5,702,716; 5,744,153; and5,990,194; wherein the suitable biodegradable thermoplastic polyester isdisclosed as a thermoplastic polymer. Examples of suitable biodegradablethermoplastic polyesters include polylactides, polyglycolides,polycaprolactones, copolymers thereof, terpolymers thereof, and anycombinations thereof. In some such embodiments, the suitablebiodegradable thermoplastic polyester is a polylactide, a polyglycolide,a copolymer thereof, a terpolymer thereof, or a combination thereof. Inone embodiment, the biodegradable thermoplastic polyester is 50/50poly(DL-lactide-co-glycolide) having a carboxy terminal group; ispresent in about 30 wt. % to about 40 wt. % of the composition; and hasan average molecular weight of about 23,000 to about 45,000.Alternatively, in another embodiment, the biodegradable thermoplasticpolyester is 75/25 poly (DL-lactide-co-glycolide) without a carboxyterminal group; is present in about 40 wt. % to about 50 wt. % of thecomposition; and has an average molecular weight of about 15,000 toabout 24,000. In further or alternative embodiments, the terminal groupsof the poly(DL-lactide-co-glycolide) are either hydroxyl, carboxyl, orester depending upon the method of polymerization. Polycondensation oflactic or glycolic acid provides a polymer with terminal hydroxyl andcarboxyl groups. Ring-opening polymerization of the cyclic lactide orglycolide monomers with water, lactic acid, or glycolic acid providespolymers with the same terminal groups. However, ring-opening of thecyclic monomers with a monofunctional alcohol such as methanol, ethanol,or 1-dodecanol provides a polymer with one hydroxyl group and one esterterminal groups. Ring-opening polymerization of the cyclic monomers witha diol such as 1,6-hexanediol or polyethylene glycol provides a polymerwith only hydroxyl terminal groups.

Since the polymer systems of thermoreversible gels dissolve morecompletely at reduced temperatures, methods of solubilization includeadding the required amount of polymer to the amount of water to be usedat reduced temperatures. Generally after wetting the polymer by shaking,the mixture is capped and placed in a cold chamber or in a thermostaticcontainer at about 0-10° C. in order to dissolve the polymer. Themixture is stirred or shaken to bring about a more rapid dissolution ofthe thermoreversible gel polymer. The auris sensory cell modulatingagent and various additives such as buffers, salts, and preservativesare subsequently added and dissolved. In some instances the aurissensory cell modulating agent and/or other pharmaceutically active agentis suspended if it is insoluble in water. The pH is modulated by theaddition of appropriate buffering agents. round window membranemucoadhesive characteristics are optionally imparted to athermoreversible gel by incorporation of round window membranemucoadhesive carbomers, such as Carbopol® 934P, to the composition(Majithiya et al, AAPS PharmSciTech (2006), 7(3), p. E1; EP0551626, bothof which is incorporated herein by reference for such disclosure).

In one embodiment are auris-acceptable pharmaceutical gel formulationswhich do not require the use of an added viscosity enhancing agent. Suchgel formulations incorporate at least one pharmaceutically acceptablebuffer. In one aspect is a gel formulation comprising an auris sensorycell modulating agent and a pharmaceutically acceptable buffer. Inanother embodiment, the pharmaceutically acceptable excipient or carrieris a gelling agent.

In other embodiments, useful auris sensory cell modulating agentauris-acceptable pharmaceutical formulations also include one or more pHadjusting agents or buffering agents to provide an endolymph orperilymph suitable pH. Suitable pH adjusting agents or buffers include,but are not limited to acetate, bicarbonate, ammonium chloride, citrate,phosphate, pharmaceutically acceptable salts thereof and combinations ormixtures thereof. Such pH adjusting agents and buffers are included inan amount required to maintain pH of the composition between a pH ofabout 5 and about 9, in one embodiment a pH between about 6.5 to about7.5, and in yet another embodiment at a pH of about 6.5, 6.6, 6.7, 6.8,6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5. In one embodiment, when one or morebuffers are utilized in the formulations of the present disclosure, theyare combined, e.g., with a pharmaceutically acceptable vehicle and arepresent in the final formulation, e.g., in an amount ranging from about0.1% to about 20%, from about 0.5% to about 10%. In certain embodimentsof the present disclosure, the amount of buffer included in the gelformulations are an amount such that the pH of the gel formulation doesnot interfere with the auris media or auris interna's natural bufferingsystem, or does not interfere with the natural pH of the endolymph orperilymph: depending on where in the cochlea the auris sensory cellmodulating agent formulation is targeted. In some embodiments, fromabout 10 μM to about 200 mM concentration of a buffer is present in thegel formulation. In certain embodiments, from about a 5 mM to about a200 mM concentration of a buffer is present. In certain embodiments,from about a 20 mM to about a 100 mM concentration of a buffer ispresent. In one embodiment is a buffer such as acetate or citrate atslightly acidic pH. In one embodiment the buffer is a sodium acetatebuffer having a pH of about 4.5 to about 6.5. In one embodiment thebuffer is a sodium citrate buffer having a pH of about 5.0 to about 8.0,or about 5.5 to about 7.0.

In an alternative embodiment, the buffer used istris(hydroxymethyl)aminomethane, bicarbonate, carbonate or phosphate atslightly basic pH. In one embodiment, the buffer is a sodium bicarbonatebuffer having a pH of about 6.5 to about 8.5, or about 7.0 to about 8.0.In another embodiment the buffer is a sodium phosphate dibasic bufferhaving a pH of about 6.0 to about 9.0.

Also described herein are controlled release formulations or devicescomprising an auris sensory cell modulating agent and a viscosityenhancing agent. Suitable viscosity-enhancing agents include by way ofexample only, gelling agents and suspending agents. In one embodiment,the enhanced viscosity formulation does not include a buffer. In otherembodiments, the enhanced viscosity formulation includes apharmaceutically acceptable buffer. Sodium chloride or other tonicityagents are optionally used to adjust tonicity, if necessary.

By way of example only, the auris-acceptable viscosity agent includehydroxypropyl methylcellulose, hydroxyethyl cellulose,polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodiumchondroitin sulfate, sodium hyaluronate. Other viscosity enhancingagents compatible with the targeted auris structure include, but are notlimited to, acacia (gum arabic), agar, aluminum magnesium silicate,sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer,carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose(MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus,dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite,lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch,wheat starch, rice starch, potato starch, gelatin, sterculia gum,xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethylcellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethylcellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose,poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline,povidone, propylene carbonate, methyl vinyl ether/maleic anhydridecopolymer (PVM/MA), poly(methoxyethyl methacrylate),poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose,hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose(CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda®(dextrose, maltodextrin and sucralose) or combinations thereof. Inspecific embodiments, the viscosity-enhancing excipient is a combinationof MCC and CMC. In another embodiment, the viscosity-enhancing agent isa combination of carboxymethylated chitosan, or chitin, and alginate.The combination of chitin and alginate with the auris sensory cellmodulating agents disclosed herein acts as a controlled releaseformulation, restricting the diffusion of the auris sensory cellmodulating agents from the formulation. Moreover, the combination ofcarboxymethylated chitosan and alginate is optionally used to assist inincreasing the permeability of the auris sensory cell modulating agentsthrough the round window membrane.

In some embodiments is an enhanced viscosity formulation, comprisingfrom about 0.1 mM and about 100 mM of an auris sensory cell modulatingagent, a pharmaceutically acceptable viscosity agent, and water forinjection, the concentration of the viscosity agent in the water beingsufficient to provide a enhanced viscosity formulation with a finalviscosity from about 100 to about 100,000 cP. In certain embodiments,the viscosity of the gel is in the range from about 100 to about 50,000cP, about 100 cP to about 1,000 cP, about 500 cP to about 1500 cP, about1000 cP to about 3000 cP, about 2000 cP to about 8,000 cP, about 4,000cP to about 50,000 cP, about 10,000 cP to about 500,000 cP, about 15,000cP to about 1,000,000 cP. In other embodiments, when an even moreviscous medium is desired, the biocompatible gel comprises at leastabout 35%, at least about 45%, at least about 55%, at least about 65%,at least about 70%, at least about 75%, or even at least about 80% or soby weight of the auris sensory cell modulating agent. In highlyconcentrated samples, the biocompatible enhanced viscosity formulationcomprises at least about 25%, at least about 35%, at least about 45%, atleast about 55%, at least about 65%, at least about 75%, at least about85%, at least about 90% or at least about 95% or more by weight of theauris sensory cell modulating agent.

In some embodiments, the viscosity of the gel formulations presentedherein are measured by any means described. For example, in someembodiments, an LVDV-II+CP Cone Plate Viscometer and a Cone SpindleCPE-40 is used to calculate the viscosity of the gel formulationdescribed herein. In other embodiments, a Brookfield (spindle and cup)viscometer is used to calculate the viscosity of the gel formulationdescribed herein. In some embodiments, the viscosity ranges referred toherein are measured at room temperature. In other embodiments, theviscosity ranges referred to herein are measured at body temperature(e.g., at the average body temperature of a healthy human).

In one embodiment, the pharmaceutically acceptable enhanced viscosityauris-acceptable formulation comprises at least one auris sensory cellmodulating agent and at least one gelling agent. Suitable gelling agentsfor use in preparation of the gel formulation include, but are notlimited to, celluloses, cellulose derivatives, cellulose ethers (e.g.,carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxymethylcellulose, hydroxypropylmethylcellulose,hydroxypropylcellulose, methylcellulose), guar gum, xanthan gum, locustbean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth,carboxyvinyl polymers, carrageenan, paraffin, petrolatum and anycombinations or mixtures thereof. In some other embodiments,hydroxypropylmethylcellulose (Methocel®) is utilized as the gellingagent. In certain embodiments, the viscosity enhancing agents describedherein are also utilized as the gelling agent for the gel formulationspresented herein.

In some embodiments, the otic therapeutic agents disclosed herein aredispensed as an auris-acceptable paint. As used herein, paints (alsoknown as film formers) are solutions comprised of a solvent, a monomeror polymer, an active agent, and optionally one or morepharmaceutically-acceptable excipients. After application to a tissue,the solvent evaporates leaving behind a thin coating comprised of themonomers or polymers, and the active agent. The coating protects activeagents and maintains them in an immobilized state at the site ofapplication. This decreases the amount of active agent which may be lostand correspondingly increases the amount delivered to the subject. Byway of non-limiting example, paints include collodions (e.g. FlexibleCollodion, USP), and solutions comprising saccharide siloxane copolymersand a cross-linking agent. Collodions are ethyl ether/ethanol solutionscontaining pyroxylin (a nitrocellulose). After application, the ethylether/ethanol solution evaporates leaving behind a thin film ofpyroxylin. In solutions comprising saccharide siloxane copolymers, thesaccharide siloxane copolymers form the coating after evaporation of thesolvent initiates the cross-linking of the saccharide siloxanecopolymers. For additional disclosures regarding paints, see Remington:The Science and Practice of Pharmacy which is hereby incorporated withrespect to this subject matter. The paints contemplated for use herein,are flexible such that they do not interfere with the propagation ofpressure waves through the ear. Further, the paints may be applied as aliquid (i.e. solution, suspension, or emulsion), a semisolid (i.e. agel, foam, paste, or jelly) or an aerosol.

In some embodiments, the otic therapeutic agents disclosed herein aredispensed as a controlled-release foam. Examples of suitable foamablecarriers for use in the compositions disclosed herein include, but arenot limited to, alginate and derivatives thereof, carboxymethylcelluloseand derivatives thereof, collagen, polysaccharides, including, forexample, dextran, dextran derivatives, pectin, starch, modified starchessuch as starches having additional carboxyl and/or carboxamide groupsand/or having hydrophilic side-chains, cellulose and derivativesthereof, agar and derivatives thereof, such as agar stabilised withpolyacrylamide, polyethylene oxides, glycol methacrylates, gelatin, gumssuch as xanthum, guar, karaya, gellan, arabic, tragacanth and locustbean gum, or combinations thereof. Also suitable are the salts of theaforementioned carriers, for example, sodium alginate. The formulationoptionally further comprises a foaming agent, which promotes theformation of the foam, including a surfactant or external propellant.Examples of suitable foaming agents include cetrimide, lecithin, soaps,silicones and the like. Commercially available surfactants such asTween® are also suitable.

In some embodiments, other gel formulations are useful depending uponthe particular auris sensory cell modulating agent, other pharmaceuticalagent or excipients/additives used, and as such are considered to fallwithin the scope of the present disclosure. For example, othercommercially-available glycerin-based gels, glycerin-derived compounds,conjugated, or crosslinked gels, matrices, hydrogels, and polymers, aswell as gelatins and their derivatives, alginates, and alginate-basedgels, and even various native and synthetic hydrogel andhydrogel-derived compounds are all expected to be useful in the aurissensory cell modulating agent formulations described herein. In someembodiments, auris-acceptable gels include, but are not limited to,alginate hydrogels SAF®-Gel (ConvaTec, Princeton, N.J.), Duoderm®Hydroactive Gel (ConvaTec), Nu-gel® (Johnson & Johnson Medical,Arlington, Tex.); Carrasyn® (V) Acemannan Hydrogel (CarringtonLaboratories, Inc., Irving, Tex.); glycerin gels Elta® Hydrogel(Swiss-American Products, Inc., Dallas, Tex.) and K-Y® Sterile (Johnson& Johnson). In further embodiments, biodegradable biocompatible gelsalso represent compounds present in auris-acceptable formulationsdisclosed and described herein.

In some formulations developed for administration to a mammal, and forcompositions formulated for human administration, the auris-acceptablegel comprises substantially all of the weight of the composition. Inother embodiments, the auris-acceptable gel comprises as much as about98% or about 99% of the composition by weight. This is desirous when asubstantially non-fluid, or substantially viscous formulation is needed.In a further embodiment, when slightly less viscous, or slightly morefluid auris-acceptable pharmaceutical gel formulations are desired, thebiocompatible gel portion of the formulation comprises at least about50% by weight, at least about 60% by weight, at least about 70% byweight, or even at least about 80% or 90% by weight of the compound. Allintermediate integers within these ranges are contemplated to fallwithin the scope of this disclosure, and in some alternativeembodiments, even more fluid (and consequently less viscous)auris-acceptable gel compositions are formulated, such as for example,those in which the gel or matrix component of the mixture comprises notmore than about 50% by weight, not more than about 40% by weight, notmore than about 30% by weight, or even those than comprise not more thanabout 15% or about 20% by weight of the composition.

Auris-Acceptable Suspending Agents

In one embodiment, at least one auris sensory cell modulating agent isincluded in a pharmaceutically acceptable enhanced viscosity formulationwherein the formulation further comprises at least one suspending agent,wherein the suspending agent assists in imparting controlled releasecharacteristics to the formulation. In some embodiments, suspendingagents also serve to increase the viscosity of the auris-acceptableauris sensory cell modulating agent formulations and compositions.

Suspending agents include, by way of example only, compounds such aspolyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12,polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, orpolyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetate copolymer(S630), sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose (hypromellose), hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylatedsorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone andthe like. In some embodiments, useful aqueous suspensions also containone or more polymers as suspending agents. Useful polymers includewater-soluble polymers such as cellulosic polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linkedcarboxyl-containing polymers.

In one embodiment, the present disclosure provides auris-acceptable gelcompositions comprising a therapeutically effective amount of an aurissensory cell modulating agent in a hydroxyethyl cellulose gel.Hydroxyethyl cellulose (HEC) is obtained as a dry powder which isreconstituted in water or an aqueous buffer solution to give the desiredviscosity (generally about 200 cps to about 30,000 cps, corresponding toabout 0.2 to about 10% HEC). In one embodiment the concentration of HECis between about 1% and about 15%, about 1% and about 2%, or about 1.5%to about 2%.

In other embodiments, the auris-acceptable formulations, including gelformulations and viscosity-enhanced formulations, further includeexcipients, other medicinal or pharmaceutical agents, carriers,adjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts, solubilizers, an antifoaming agent,an antioxidant, a dispersing agent, a wetting agent, a surfactant, andcombinations thereof.

Auris-Acceptable Actinic Radiation Curable Gel

In other embodiments, the gel is an actinic radiation curable gel, suchthat following administration to or near the targeted auris structure,use of actinic radiation (or light, including UV light, visible light,or infrared light) the desired gel properties are formed. By way ofexample only, fiber optics are used to provide the actinic radiation soas to form the desired gel properties. In some embodiments, the fiberoptics and the gel administration device form a single unit. In otherembodiments, the fiber optics and the gel administration device areprovided separately.

Auris-Acceptable Solvent Release Gel

In some embodiments, the gel is a solvent release gel such that thedesired gel properties are formed after administration to or near thetargeted auris structure, that is, as the solvent in the injected gelformulation diffuses out the gel, a gel having the desired gelproperties is formed. For example, a formulation that comprises sucroseacetate isobutyrate, a pharmaceutically acceptable solvent, one or moreadditives, and the auris sensory cell modulating agent is administeredat or near the round window membrane: diffusion of the solvent out ofthe injected formulation provides a depot having the desired gelproperties. For example, use of a water soluble solvent provides a highviscosity depot when the solvent diffuses rapidly out of the injectedformulation. On the other hand, use of a hydrophobic solvent (e.g.,benzyl benzoate) provides a less viscous depot. One example of anauris-acceptable solvent release gel formulation is the SABER™ DeliverySystem marketed by DURECT Corporation.

Auris-Acceptable In Situ Forming Spongy Material

Also contemplated within the scope of the embodiments is the use of aspongy material, formed in situ in the auris interna or auris media. Insome embodiments, the spongy material is formed from hyaluronic acid orits derivatives. The spongy material is impregnated with a desired aurissensory cell modulating agent and placed within the auris media so as toprovide controlled release of the auris sensory cell modulating agentwithin the auris media, or in contact with the round window membrane soas to provide controlled release of the auris sensory cell modulatingagent into the auris interna. In some embodiments, the spongy materialis biodegradable.

Round Window Membrane Mucoadhesives

Also contemplated within the scope of the embodiments is the addition ofa round window membrane mucoadhesive with the auris sensory cellmodulating agent formulations and compositions and devices disclosedherein. The term ‘mucoadhesion’ is used for materials that bind to themucin layer of a biological membrane, such as the external membrane ofthe 3-layered round window membrane. To serve as round window membranemucoadhesive polymers, the polymers possess some general physiochemicalfeatures such as predominantly anionic hydrophilicity with numeroushydrogen bond forming groups, suitable surface property for wettingmucus/mucosal tissue surfaces or sufficient flexibility to penetrate themucus network.

Round window membrane mucoadhesive agents that are used with theauris-acceptable formulations include, but are not limited to, at leastone soluble polyvinylpyrrolidone polymer (PVP); a water-swellable, butwater-insoluble, fibrous, cross-linked carboxy-functional polymer; acrosslinked poly(acrylic acid) (e.g. Carbopol® 947P); a carbomerhomopolymer; a carbomer copolymer; a hydrophilic polysaccharide gum,maltodextrin, a cross-linked alginate gum gel, a water-dispersiblepolycarboxylated vinyl polymer, at least two particulate componentsselected from the group consisting of titanium dioxide, silicon dioxide,and clay, or a mixture thereof. The round window membrane mucoadhesiveagent is optionally used in combination with an auris-acceptableviscosity increasing excipient, or used alone to increase theinteraction of the composition with the mucosal layer target oticcomponent. In one non-limiting example, the mucoadhesive agent ismaltodextrin. In some embodiments, the mucoadhesive agent is an alginategum. When used, the round window membrane mucoadhesive characterimparted to the composition is at a level that is sufficient to deliveran effective amount of the auris sensory cell modulating agentcomposition to, for example, the mucosal layer of round window membraneor the crista fenestrae cochleae in an amount that coats the mucosalmembrane, and thereafter deliver the composition to the affected areas,including by way of example only, the vestibular and/or cochlearstructures of the auris interna. When used, the mucoadhesivecharacteristics of the compositions provided herein are determined, andusing this information (along with the other teachings provided herein),the appropriate amounts are determined. One method for determiningsufficient mucoadhesiveness includes monitoring changes in theinteraction of the composition with a mucosal layer, including but notlimited to measuring changes in residence or retention time of thecomposition in the absence and presence of the mucoadhesive excipient.

Mucoadhesive agents have been described, for example, in U.S. Pat. Nos.6,638,521, 6,562,363, 6,509,028, 6,348,502, 6,319,513, 6,306,789,5,814,330, and 4,900,552, each of which is hereby incorporated byreference for such disclosure.

In another non-limiting example, a mucoadhesive agent is, for example,at least two particulate components selected from titanium dioxide,silicon dioxide, and clay, wherein the composition is not furtherdiluted with any liquid prior to administration and the level of silicondioxide, if present, is from about 3% to about 15%, by weight of thecomposition. Silicon dioxide, if present, includes fumed silicondioxide, precipitated silicon dioxide, coacervated silicon dioxide, gelsilicon dioxide, and mixtures thereof. Clay, if present, includes kaolinminerals, serpentine minerals, smectites, illite or a mixture thereof.For example, clay includes laponite, bentonite, hectorite, saponite,montmorillonites or a mixture thereof.

In one non-limiting example, the round window membrane mucoadhesiveagent is maltodextrin. Maltodextrin is a carbohydrate produced by thehydrolysis of starch that is optionally derived from corn, potato, wheator other plant products. Maltodextrin is optionally used either alone orin combination with other round window membrane mucoadhesive agents toimpart mucoadhesive characteristics on the compositions disclosedherein. In one embodiment, a combination of maltodextrin and a carbopolpolymer are used to increase the round window membrane mucoadhesivecharacteristics of the compositions or devices disclosed herein.

In another embodiment, the round window membrane mucoadhesive agent isan alkyl-glycoside and/or a saccharide alkyl ester. As used herein, an“alkyl-glycoside” means a compound comprising any hydrophilic saccharide(e.g. sucrose, maltose, or glucose) linked to a hydrophobic alkyl. Insome embodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl-glycoside comprises a sugar linked toa hydrophobic alkyl (e.g., an alkyl comprising about 6 to about 25carbon atoms) by an amide linkage, an amine linkage, a carbamatelinkage, an ether linkage, a thioether linkage, an ester linkage, athioester linkage, a glycosidic linkage, a thioglycosidic linkage,and/or a ureide linkage. In some embodiments, the round window membranemucoadhesive agent is a hexyl-, heptyl-, octyl-, nonyl-, decyl-,undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-, hexadecyl-,heptadecyl-, and octadecyl α- or β-D-maltoside; hexyl-, heptyl-, octyl-,nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-, tetradecyl, pentadecyl-,hexadecyl-, heptadecyl-, and octadecyl α- or β-D-glucoside; hexyl-,heptyl-, octyl-, nonyl-, decyl-, undecyl-, dodecyl-, tridecyl-,tetradecyl, pentadecyl-, hexadecyl-, heptadecyl-, and octadecyl α- orβ-D-sucroside; hexyl-, heptyl-, octyl-, dodecyl-, tridecyl-, andtetradecyl-β-D-thiomaltoside; dodecyl maltoside; heptyl- oroctyl-1-thio-α- or β-D-glucopyranoside; alkyl thiosucroses; alkylmaltotriosides; long chain aliphatic carbonic acid amides of sucroseβ-amino-alkyl ethers; derivatives of palatinose or isomaltamine linkedby an amide linkage to an alkyl chain and derivatives of isomaltaminelinked by urea to an alkyl chain; long chain aliphatic carbonic acidureides of sucrose β-amino-alkyl ethers and long chain aliphaticcarbonic acid amides of sucrose β3-amino-alkyl ethers. In someembodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl glycoside is maltose, sucrose,glucose, or a combination thereof linked by a glycosidic linkage to analkyl chain of 9-16 carbon atoms (e.g., nonyl-, decyl-, dodecyl- andtetradecyl sucroside; nonyl-, decyl-, dodecyl- and tetradecyl glucoside;and nonyl-, decyl-, dodecyl- and tetradecyl maltoside). In someembodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl glycoside is dodecylmaltoside,tridecylmaltoside, and tetradecylmaltoside.

In some embodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl-glycoside is a disaccharide with atleast one glucose. In some embodiments, the auris acceptable penetrationenhancer is a surfactant comprisingα-D-glucopyranosyl-β-glycopyranoside,n-Dodecyl-4-O-α-D-glucopyranosyl-β-glycopyranoside, and/orn-tetradecyl-4-O-α-D-glucopyranosyl-β-glycopyranoside. In someembodiments, the round window membrane mucoadhesive agent is analkyl-glycoside wherein the alkyl-glycoside has a critical miscelleconcentration (CMC) of less than about 1 mM in pure water or in aqueoussolutions. In some embodiments, the round window membrane mucoadhesiveagent is an alkyl-glycoside wherein an oxygen atom within thealkyl-glycoside is substituted with a sulfur atom. In some embodiments,the round window membrane mucoadhesive agent is an alkyl-glycosidewherein the alkylglycoside is the β anomer. In some embodiments, theround window membrane mucoadhesive agent is an alkyl-glycoside whereinthe alkylglycoside comprises 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.5%, or 99.9% of the β anomer.

Auris-Acceptable Controlled Release Particles

Auris sensory cell modulating agents and/or other pharmaceutical agentsdisclosed herein are optionally incorporated within controlled releaseparticles, lipid complexes, liposomes, nanoparticles, microparticles,microspheres, coacervates, nanocapsules or other agents which enhance orfacilitate the localized delivery of the auris sensory cell modulatingagent. In some embodiments, a single enhanced viscosity formulation isused, in which at least one auris sensory cell modulating agent ispresent, while in other embodiments, a pharmaceutical formulation thatcomprises a mixture of two or more distinct enhanced viscosityformulations is used, in which at least one auris sensory cellmodulating agent is present. In some embodiments, combinations of sols,gels and/or biocompatible matrices is also employed to provide desirablecharacteristics of the controlled release auris sensory cell modulatingagent compositions or formulations. In certain embodiments, thecontrolled release auris sensory cell modulating agent formulations orcompositions are cross-linked by one or more agents to alter or improvethe properties of the composition.

Examples of microspheres relevant to the pharmaceutical formulationsdisclosed herein include: Luzzi, L. A., J. Pharm. Psy. 59:1367 (1970);U.S. Pat. No. 4,530,840; Lewis, D. H., “Controlled Release of BioactiveAgents from Lactides/Glycolide Polymers” in Biodegradable Polymers asDrug Delivery Systems, Chasin, M. and Langer, R., eds., Marcel Decker(1990); U.S. Pat. No. 4,675,189; Beck et al., “Poly(lactic acid) andPoly(lactic acid-co-glycolic acid) Contraceptive Delivery Systems,” inLong Acting Steroid Contraception, Mishell, D. R., ed., Raven Press(1983); U.S. Pat. No. 4,758,435; U.S. Pat. No. 3,773,919; U.S. Pat. No.4,474,572. Examples of protein therapeutics formulated as microspheresinclude: U.S. Pat. No. 6,458,387; U.S. Pat. No. 6,268,053; U.S. Pat. No.6,090,925; U.S. Pat. No. 5,981,719; and U.S. Pat. No. 5,578,709, and areherein incorporated by reference for such disclosure.

Microspheres usually have a spherical shape, although irregularly-shapedmicroparticles are possible. Microspheres may vary in size, ranging fromsubmicron to 1000 micron diameters. Microspheres suitable for use withthe auris-acceptable formulations disclosed herein are submicron to 250micron diameter microspheres, allowing administration by injection witha standard gauge needle. The auris-acceptable microspheres are preparedby any method which produces microspheres in a size range acceptable foruse in an injectable composition. Injection is optionally accomplishedwith standard gauge needles used for administering liquid compositions.

Suitable examples of polymeric matrix materials for use in theauris-acceptable controlled release particles herein includepoly(glycolic acid), poly-d,1-lactic acid, poly-1-lactic acid,copolymers of the foregoing, poly(aliphatic carboxylic acids),copolyoxalates, polycaprolactone, polydioxonene, poly(orthocarbonates),poly(acetals), poly(lactic acid-caprolactone), polyorthoesters,poly(glycolic acid-caprolactone), polydioxonene, polyanhydrides,polyphosphazines, and natural polymers including albumin, casein, andsome waxes, such as, glycerol mono- and distearate, and the like.Various commercially available poly (lactide-co-glycolide) materials(PLGA) are optionally used in the method disclosed herein. For example,poly (d,1-lactic-co-glycolic acid) is commercially available fromBoehringer-Ingelheim as RESOMER RG 503H. This product has a mole percentcomposition of 50% lactide and 50% glycolide. These copolymers areavailable in a wide range of molecular weights and ratios of lactic acidto glycolic acid. One embodiment includes the use of the polymerpoly(d,1-lactide-co-glycolide). The molar ratio of lactide to glycolidein such a copolymer includes the range of from about 95:5 to about50:50.

The molecular weight of the polymeric matrix material is of someimportance. The molecular weight should be high enough so that it formssatisfactory polymer coatings, i.e., the polymer should be a good filmformer. Usually, a satisfactory molecular weight is in the range of5,000 to 500,000 daltons. The molecular weight of a polymer is alsoimportant from the point of view that molecular weight influences thebiodegradation rate of the polymer. For a diffusional mechanism of drugrelease, the polymer should remain intact until all of the drug isreleased from the microparticles and then degrade. The drug is alsoreleased from the microparticles as the polymeric excipient bioerodes.By an appropriate selection of polymeric materials a microsphereformulation is made such that the resulting microspheres exhibit bothdiffusional release and biodegradation release properties. This isuseful in affording multiphasic release patterns.

A variety of methods are known by which compounds are encapsulated inmicrospheres. In these methods, the auris sensory cell modulating agentis generally dispersed or emulsified, using stirrers, agitators, orother dynamic mixing techniques, in a solvent containing a wall-formingmaterial. Solvent is then removed from the microspheres, and thereafterthe microsphere product is obtained.

In one embodiment, controlled release auris sensory cell modulatingagent formulations are made through the incorporation of the aurissensory cell modulating agents and/or other pharmaceutical agents intoethylene-vinyl acetate copolymer matrices. (See U.S. Pat. No. 6,083,534,incorporated herein for such disclosure). In another embodiment, aurissensory cell modulating agents are incorporated into poly(lactic-glycolic acid) or poly-L-lactic acid microspheres. Id. In yetanother embodiment, the auris sensory cell modulating agents areencapsulated into alginate microspheres. (See U.S. Pat. No. 6,036,978,incorporated herein for such disclosure). Biocompatiblemethacrylate-based polymers to encapsulate the auris sensory cellmodulating agent compounds or compositions are optionally used in theformulations and methods disclosed herein. A wide range ofmethacrylate-based polymer systems are commercially available, such asthe EUDRAGIT polymers marketed by Evonik. One useful aspect ofmethacrylate polymers is that the properties of the formulation arevaried by incorporating various co-polymers. For example, poly(acrylicacid-co-methylmethacrylate) microparticles exhibit enhanced mucoadhesionproperties as the carboxylic acid groups in the poly(acrylic acid) formhydrogen bonds with mucin (Park et al, Pharm. Res. (1987) 4(6):457-464).Variation of the ratio between acrylic acid and methylmethacrylatemonomers serves to modulate the properties of the co-polymer.Methacrylate-based microparticles have also been used in proteintherapeutic formulations (Naha et al, Journal of Microencapsulation 4Feb., 2008 (online publication)). In one embodiment, the enhancedviscosity auris-acceptable formulations described herein comprises aurissensory cell modulating agent microspheres wherein the microspheres areformed from a methacrylate polymer or copolymer. In an additionalembodiment, the enhanced viscosity formulation described hereincomprises auris sensory cell modulating agent microspheres wherein themicrospheres are mucoadhesive. Other controlled release systems,including incorporation or deposit of polymeric materials or matricesonto solid or hollow spheres containing auris sensory cell modulatingagents, are also explicitly contemplated within the embodimentsdisclosed herein. The types of controlled release systems availablewithout significantly losing activity of the auris sensory cellmodulating agent are determined using the teachings, examples, andprinciples disclosed herein

An example of a conventional microencapsulation process forpharmaceutical preparations is shown in U.S. Pat. No. 3,737,337,incorporated herein by reference for such disclosure. The auris sensorycell modulating agent substances to be encapsulated or embedded aredissolved or dispersed in the organic solution of the polymer (phase A),using conventional mixers, including (in the preparation of dispersion)vibrators and high-speed stirrers, etc. The dispersion of phase (A),containing the core material in solution or in suspension, is carriedout in the aqueous phase (B), again using conventional mixers, such ashigh-speed mixers, vibration mixers, or even spray nozzles, in whichcase the particle size of the microspheres will be determined not onlyby the concentration of phase (A), but also by the emulsate ormicrosphere size. With conventional techniques for themicroencapsulation of auris sensory cell modulating agents, themicrospheres form when the solvent containing an active agent and apolymer is emulsified or dispersed in an immiscible solution bystirring, agitating, vibrating, or some other dynamic mixing technique,often for a relatively long period of time.

Methods for the construction of microspheres are also described in U.S.Pat. No. 4,389,330, and U.S. Pat. No. 4,530,840, incorporated herein byreference for such disclosure. The desired auris sensory cell modulatingagent is dissolved or dispersed in an appropriate solvent. To theagent-containing medium is added the polymeric matrix material in anamount relative to the active ingredient which gives a product of thedesired loading of active agent. Optionally, all of the ingredients ofthe auris sensory cell modulating agent microsphere product can beblended in the solvent medium together. Suitable solvents for the agentand the polymeric matrix material include organic solvents such asacetone, halogenated hydrocarbons such as chloroform, methylene chlorideand the like, aromatic hydrocarbon compounds, halogenated aromatichydrocarbon compounds, cyclic ethers, alcohols, ethyl acetate and thelike.

The mixture of ingredients in the solvent is emulsified in acontinuous-phase processing medium; the continuous-phase medium beingsuch that a dispersion of microdroplets containing the indicatedingredients is formed in the continuous-phase medium. Naturally, thecontinuous-phase processing medium and the organic solvent must beimmiscible, and includes water although nonaqueous media such as xyleneand toluene and synthetic oils and natural oils are optionally used.Optionally, a surfactant is added to the continuous-phase processingmedium to prevent the microparticles from agglomerating and to controlthe size of the solvent microdroplets in the emulsion. A preferredsurfactant-dispersing medium combination is a 1 to 10 wt. % poly (vinylalcohol) in water mixture. The dispersion is formed by mechanicalagitation of the mixed materials. An emulsion is optionally formed byadding small drops of the active agent-wall forming material solution tothe continuous phase processing medium. The temperature during theformation of the emulsion is not especially critical but influences thesize and quality of the microspheres and the solubility of the drug inthe continuous phase. It is desirable to have as little of the agent inthe continuous phase as possible. Moreover, depending on the solvent andcontinuous-phase processing medium employed, the temperature must not betoo low or the solvent and processing medium will solidify or theprocessing medium will become too viscous for practical purposes, or toohigh that the processing medium will evaporate, or that the liquidprocessing medium will not be maintained. Moreover, the temperature ofthe medium cannot be so high that the stability of the particular agentbeing incorporated in the microspheres is adversely affected.Accordingly, the dispersion process is conducted at any temperaturewhich maintains stable operating conditions, which preferred temperaturebeing about 15° C. to 60° C., depending upon the drug and excipientselected.

The dispersion which is formed is a stable emulsion and from thisdispersion the organic solvent immiscible fluid is optionally partiallyremoved in the first step of the solvent removal process. The solvent isremoved by techniques such as heating, the application of a reducedpressure or a combination of both. The temperature employed to evaporatesolvent from the microdroplets is not critical, but should not be thathigh that it degrades the auris sensory cell modulating agent employedin the preparation of a given microparticle, nor should it be so high asto evaporate solvent at such a rapid rate to cause defects in the wallforming material. Generally, from 5 to 75%, of the solvent is removed inthe first solvent removal step.

After the first stage, the dispersed microparticles in the solventimmiscible fluid medium are isolated from the fluid medium by anyconvenient means of separation. Thus, for example, the fluid is decantedfrom the microsphere or the microsphere suspension is filtered. Stillother, various combinations of separation techniques are used ifdesired.

Following the isolation of the microspheres from the continuous-phaseprocessing medium, the remainder of the solvent in the microspheres isremoved by extraction. In this step, the microspheres are suspended inthe same continuous-phase processing medium used in step one, with orwithout surfactant, or in another liquid. The extraction medium removesthe solvent from the microspheres and yet does not dissolve themicrospheres. During the extraction, the extraction medium withdissolved solvent is optionally removed and replaced with freshextraction medium. This is best done on a continual basis. The rate ofextraction medium replenishment of a given process is a variable whichis determined at the time the process is performed and, therefore, noprecise limits for the rate must be predetermined. After the majority ofthe solvent has been removed from the microspheres, the microspheres aredried by exposure to air or by other conventional drying techniques suchas vacuum drying, drying over a desiccant, or the like. This process isvery efficient in encapsulating the auris sensory cell modulating agentsince core loadings of up to 80 wt. %, preferably up to 60 wt. % areobtained.

Alternatively, controlled release microspheres containing an aurissensory cell modulating agent is prepared through the use of staticmixers. Static or motionless mixers consist of a conduit or tube inwhich is received a number of static mixing agents. Static mixersprovide homogeneous mixing in a relatively short length of conduit, andin a relatively short period of time. With static mixers, the fluidmoves through the mixer, rather than some part of the mixer, such as ablade, moving through the fluid.

A static mixer is optionally used to create an emulsion. When using astatic mixer to form an emulsion, several factors determine emulsionparticle size, including the density and viscosity of the varioussolutions or phases to be mixed, volume ratio of the phases, interfacialtension between the phases, static mixer parameters (conduit diameter;length of mixing element; number of mixing elements), and linearvelocity through the static mixer. Temperature is a variable because itaffects density, viscosity, and interfacial tension. The controllingvariables are linear velocity, sheer rate, and pressure drop per unitlength of static mixer.

In order to create microspheres containing an auris sensory cellmodulating agent using a static mixer process, an organic phase and anaqueous phase are combined. The organic and aqueous phases are largelyor substantially immiscible, with the aqueous phase constituting thecontinuous phase of the emulsion. The organic phase includes an aurissensory cell modulating agent as well as a wall-forming polymer orpolymeric matrix material. The organic phase is prepared by dissolvingan auris sensory cell modulating agent in an organic or other suitablesolvent, or by forming a dispersion or an emulsion containing the aurissensory cell modulating agent. The organic phase and the aqueous phaseare pumped so that the two phases flow simultaneously through a staticmixer, thereby forming an emulsion which comprises microspherescontaining the auris sensory cell modulating agent encapsulated in thepolymeric matrix material. The organic and aqueous phases are pumpedthrough the static mixer into a large volume of quench liquid to extractor remove the organic solvent. Organic solvent is optionally removedfrom the microspheres while they are washing or being stirred in thequench liquid. After the microspheres are washed in a quench liquid,they are isolated, as through a sieve, and dried.

In one embodiment, microspheres are prepared using a static mixer. Theprocess is not limited to the solvent extraction technique discussedabove, but is used with other encapsulation techniques. For example, theprocess is optionally used with a phase separation encapsulationtechnique. To do so, an organic phase is prepared that comprises anauris sensory cell modulating agent suspended or dispersed in a polymersolution. The non-solvent second phase is free from solvents for thepolymer and active agent. A preferred non-solvent second phase issilicone oil. The organic phase and the non-solvent phase are pumpedthrough a static mixer into a non-solvent quench liquid, such asheptane. The semi-solid particles are quenched for complete hardeningand washing. The process of microencapsulation includes spray drying,solvent evaporation, a combination of evaporation and extraction, andmelt extrusion.

In another embodiment, the microencapsulation process involves the useof a static mixer with a single solvent. This process is described indetail in U.S. application Ser. No. 08/338,805, herein incorporated byreference for such disclosure. An alternative process involves the useof a static mixer with co-solvents. In this process, biodegradablemicrospheres comprising a biodegradable polymeric binder and an aurissensory cell modulating agent are prepared, which comprises a blend ofat least two substantially non-toxic solvents, free of halogenatedhydrocarbons to dissolve both the agent and the polymer. The solventblend containing the dissolved agent and polymer is dispersed in anaqueous solution to form droplets. The resulting emulsion is then addedto an aqueous extraction medium preferably containing at least one ofthe solvents of the blend, whereby the rate of extraction of eachsolvent is controlled, whereupon the biodegradable microspherescontaining the pharmaceutically active agent are formed. This processhas the advantage that less extraction medium is required because thesolubility of one solvent in water is substantially independent of theother and solvent selection is increased, especially with solvents thatare particularly difficult to extract.

Nanoparticles are also contemplated for use with the auris sensory cellmodulating agents disclosed herein. Nanoparticles are materialstructures of about 100 nm or less in size. One use of nanoparticles inpharmaceutical formulations is the formation of suspensions as theinteraction of the particle surface with solvent is strong enough toovercome differences in density. Nanoparticle suspensions are sterilizedas the nanoparticles are small enough to be subjected to sterilizingfiltration (see, e.g., U.S. Pat. No. 6,139,870, herein incorporated byreference for such disclosure). Nanoparticles comprise at least onehydrophobic, water-insoluble and water-indispersible polymer orcopolymer emulsified in a solution or aqueous dispersion of surfactants,phospholipids or fatty acids. The auris sensory cell modulating agent isoptionally introduced with the polymer or the copolymer into thenanoparticles.

Lipid nanocapsules as controlled release structures, as well forpenetrating the round window membrane and reaching auris interna and/orauris media targets, is also contemplated herein. Lipid nanocapsules areoptionally formed by emulsifying capric and caprylic acid triglycerides(Labrafac WL 1349; avg. mw 512), soybean lecithin (LIPOID® S75-3; 69%phosphatidylcholine and other phospholipids), surfactant (for example,Solutol HS15), a mixture of polyethylene glycol 660 hydroxystearate andfree polyethylene glycol 660; NaCl and water. The mixture is stirred atroom temperature to obtain an oil emulsion in water. After progressiveheating at a rate of 4° C./min under magnetic stirring, a short intervalof transparency should occur close to 70° C., and the inverted phase(water droplets in oil) obtained at 85° C. Three cycles of cooling andheating is then applied between 85° C. and 60° C. at the rate of 4°C./min, and a fast dilution in cold water at a temperature close to 0°C. to produce a suspension of nanocapsules. To encapsulate the aurissensory cell modulating agents, the agent is optionally added just priorto the dilution with cold water.

Auris sensory cell modulating agents are also inserted into the lipidnanocapsules by incubation for 90 minutes with an aqueous micellarsolution of the auris active agent. The suspension is then vortexedevery 15 minutes, and then quenched in an ice bath for 1 minute.

Suitable auris-acceptable surfactants are, by way of example, cholicacid or taurocholic acid salts. Taurocholic acid, the conjugate formedfrom cholic acid and taurine, is a fully metabolizable sulfonic acidsurfactant. An analog of taurocholic acid, tauroursodeoxycholic acid(TUDCA), is a naturally occurring bile acid and is a conjugate oftaurine and ursodeoxycholic acid (UDCA). Other naturally occurringanionic (e.g., galactocerebroside sulfate), neutral (e.g.,lactosylceramide) or zwitterionic surfactants (e.g., sphingomyelin,phosphatidyl choline, palmitoyl carnitine) are optionally used toprepare nanoparticles.

The auris-acceptable phospholipids are chosen, by way of example, fromnatural, synthetic or semi-synthetic phospholipids; lecithins(phosphatidylcholine) such as, for example, purified egg or soyalecithins (lecithin E100, lecithin E80 and phospholipons, for examplephospholipon 90), phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol,dipalmitoylphosphatidylcholine, dipalmitoylglycerophosphatidylcholine,dimyristoylphosphatidylcholine, distearoylphosphatidylcholine andphosphatidic acid or mixtures thereof are used more particularly.

Fatty acids for use with the auris-acceptable formulations are chosenfrom, by way of example, lauric acid, mysristic acid, palmitic acid,stearic acid, isostearic acid, arachidic acid, behenic acid, oleic acid,myristoleic acid, palmitoleic acid, linoleic acid, alpha-linoleic acid,arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoicacid, and the like.

Suitable auris-acceptable surfactants are selected from known organicand inorganic pharmaceutical excipients. Such excipients include variouspolymers, low molecular weight oligomers, natural products, andsurfactants. Preferred surface modifiers include nonionic and ionicsurfactants. Two or more surface modifiers are used in combination.

Representative examples of auris-acceptable surfactants include cetylpyridinium chloride, gelatin, casein, lecithin (phosphatides), dextran,glycerol, gum acacia, cholesterol, tragacanth, stearic acid, calciumstearate, glycerol monostearate, cetostearyl alcohol, cetomacrogolemulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers,polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fattyacid esters; dodecyl trimethyl ammonium bromide,polyoxyethylenestearates, colloidal silicon dioxide, phosphates, sodiumdodecylsulfate, carboxymethylcellulose calcium, hydroxypropyl cellulose(HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose (HPMC),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethyl-cellulose phthalate,noncrystalline cellulose, magnesium aluminum silicate, triethanolamine,polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol, superione, and triton),poloxamers, poloxamines, a charged phospholipid such as dimyristoylphophatidyl glycerol, dioctylsulfosuccinate (DOSS); Tetronic® 1508,dialkylesters of sodium sulfosuccinic acid, Duponol P, Tritons X-200,Crodestas F-110, p-isononylphenoxypoly-(glycidol), Crodestas SL-40(Croda, Inc.); and SA9OHCO, which is C₁₈H₃₇CH₂ (CON(CH₃)—CH₂ (CHOH)₄(CH₂OH)₂ (Eastman Kodak Co.); decanoyl-N-methylglucamide; n-decylβ-D-glucopyranoside; n-decyl β-D-maltopyranoside; n-dodecylβ-D-glucopyranoside; n-dodecyl β-D-maltoside;heptanoyl-N-methylglucamide; n-heptyl-β-D-glucopyranoside; n-heptylβ-D-thioglucoside; n-hexyl β-D-glucopyranoside;nonanoyl-N-methylglucamide; n-noyl β-D-glucopyranoside;octanoyl-N-methylglucamide; n-octyl-β-D-glucopyranoside; octylβ-D-thioglucopyranoside; and the like. Most of these surfactants areknown pharmaceutical excipients and are described in detail in theHandbook of Pharmaceutical Excipients, published jointly by the AmericanPharmaceutical Association and The Pharmaceutical Society of GreatBritain (The Pharmaceutical Press, 1986), specifically incorporated byreference for such disclosure.

The hydrophobic, water-insoluble and water-indispersible polymer orcopolymer may be chosen from biocompatible and biodegradable polymers,for example lactic or glycolic acid polymers and copolymers thereof, orpolylactic/polyethylene (or polypropylene) oxide copolymers, preferablywith molecular weights of between 1000 and 200,000, polyhydroxybutyricacid polymers, polylactones of fatty acids containing at least 12 carbonatoms, or polyanhydrides.

The nanoparticles may be obtained by coacervation, or by the techniqueof evaporation of solvent, from an aqueous dispersion or solution ofphospholipids and of an oleic acid salt into which is added animmiscible organic phase comprising the active principle and thehydrophobic, water-insoluble and water-indispersible polymer orcopolymer. The mixture is pre-emulsified and then subjected tohomogenization and evaporation of the organic solvent to obtain anaqueous suspension of very small-sized nanoparticles.

A variety of methods are optionally employed to fabricate the aurissensory cell modulating agent nanoparticles that are within the scope ofthe embodiments. These methods include vaporization methods, such asfree jet expansion, laser vaporization, spark erosion, electro explosionand chemical vapor deposition; physical methods involving mechanicalattrition (e.g., “pearlmilling” technology, Elan Nanosystems), supercritical CO2 and interfacial deposition following solvent displacement.In one embodiment, the solvent displacement method is used. The size ofnanoparticles produced by this method is sensitive to the concentrationof polymer in the organic solvent; the rate of mixing; and to thesurfactant employed in the process. Continuous flow mixers provide thenecessary turbulence to ensure small particle size. One type ofcontinuous flow mixing device that is optionally used to preparenanoparticles has been described (Hansen et al J Phys Chem 92, 2189-96,1988). In other embodiments, ultrasonic devices, flow throughhomogenizers or supercritical CO2 devices may be used to preparenanoparticles.

If suitable nanoparticle homogeneity is not obtained on directsynthesis, then size-exclusion chromatography is used to produce highlyuniform drug-containing particles that are freed of other componentsinvolved in their fabrication. Size-exclusion chromatography (SEC)techniques, such as gel-filtration chromatography, is used to separateparticle-bound auris sensory cell modulating agent or otherpharmaceutical compound from free auris sensory cell modulating agent orother pharmaceutical compound, or to select a suitable size range ofauris sensory cell modulating agent-containing nanoparticles. VariousSEC media, such as Superdex 200, Superose 6, Sephacryl 1000 arecommercially available and are employed for the size-based fractionationof such mixtures. Additionally, nanoparticles are optionally purified bycentrifugation, membrane filtration and by use of other molecularsieving devices, crosslinked gels/materials and membranes.

Auris-Acceptable Cyclodextrin and Other Stabilizing Formulations

In a specific embodiment, the auris-acceptable formulationsalternatively comprises a cyclodextrin. Cyclodextrins are cyclicoligosaccharides containing 6, 7, or 8 glucopyranose units, referred toas α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin respectively.Cyclodextrins have a hydrophilic exterior, which enhances water-soluble,and a hydrophobic interior which forms a cavity. In an aqueousenvironment, hydrophobic portions of other molecules often enter thehydrophobic cavity of cyclodextrin to form inclusion compounds.Additionally, cyclodextrins are also capable of other types ofnonbonding interactions with molecules that are not inside thehydrophobic cavity. Cyclodextrins have three free hydroxyl groups foreach glucopyranose unit, or 18 hydroxyl groups on α-cyclodextrin, 21hydroxyl groups on β-cyclodextrin, and 24 hydroxyl groups onγ-cyclodextrin. One or more of these hydroxyl groups can be reacted withany of a number of reagents to form a large variety of cyclodextrinderivatives, including hydroxypropyl ethers, sulfonates, andsulfoalkylethers. Shown below is the structure of β-cyclodextrin and thehydroxypropyl-β-cyclodextrin (HPβCD).

In some embodiments, the use of cyclodextrins in the pharmaceuticalcompositions described herein improves the solubility of the drug.Inclusion compounds are involved in many cases of enhanced solubility;however other interactions between cyclodextrins and insoluble compoundsalso improves solubility. Hydroxypropyl-β-cyclodextrin (HPβCD) iscommercially available as a pyrogen free product. It is a nonhygroscopicwhite powder that readily dissolves in water. HPβCD is thermally stableand does not degrade at neutral pH. Thus, cyclodextrins improve thesolubility of a therapeutic agent in a composition or formulation.Accordingly, in some embodiments, cyclodextrins are included to increasethe solubility of the auris-acceptable auris sensory cell modulatingagents within the formulations described herein. In other embodiments,cyclodextrins in addition serve as controlled release excipients withinthe formulations described herein.

By way of example only, cyclodextrin derivatives for use includeα-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethylβ-cyclodextrin, hydroxypropyl γ-cyclodextrin, sulfated β-cyclodextrin,sulfated α-cyclodextrin, sulfobutyl ether β-cyclodextrin.

The concentration of the cyclodextrin used in the compositions andmethods disclosed herein varies according to the physiochemicalproperties, pharmacokinetic properties, side effect or adverse events,formulation considerations, or other factors associated with thetherapeutically active agent, or a salt or prodrug thereof, or with theproperties of other excipients in the composition. Thus, in certaincircumstances, the concentration or amount of cyclodextrin used inaccordance with the compositions and methods disclosed herein will vary,depending on the need. When used, the amount of cyclodextrins needed toincrease solubility of the auris sensory cell modulating agent and/orfunction as a controlled release excipient in any of the formulationsdescribed herein is selected using the principles, examples, andteachings described herein.

Other stabilizers that are useful in the auris-acceptable formulationsdisclosed herein include, for example, fatty acids, fatty alcohols,alcohols, long chain fatty acid esters, long chain ethers, hydrophilicderivatives of fatty acids, polyvinyl pyrrolidones, polyvinyl ethers,polyvinyl alcohols, hydrocarbons, hydrophobic polymers,moisture-absorbing polymers, and combinations thereof. In someembodiments, amide analogues of stabilizers are also used. In furtherembodiments, the chosen stabilizer changes the hydrophobicity of theformulation (e.g., oleic acid, waxes), or improves the mixing of variouscomponents in the formulation (e.g., ethanol), controls the moisturelevel in the formula (e.g., PVP or polyvinyl pyrrolidone), controls themobility of the phase (substances with melting points higher than roomtemperature such as long chain fatty acids, alcohols, esters, ethers,amides etc. or mixtures thereof; waxes), and/or improves thecompatibility of the formula with encapsulating materials (e.g., oleicacid or wax). In another embodiment some of these stabilizers are usedas solvents/co-solvents (e.g., ethanol). In other embodiments,stabilizers are present in sufficient amounts to inhibit the degradationof the auris sensory cell modulating agent. Examples of such stabilizingagents, include, but are not limited to: (a) about 0.5% to about 2% w/vglycerol, (b) about 0.1% to about 1% w/v methionine, (c) about 0.1% toabout 2% w/v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e)about 0.01% to about 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/vpolysorbate 80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h)arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l)pentosan polysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

Additional useful auris sensory cell modulating agent auris-acceptableformulations include one or more anti-aggregation additives to enhancestability of auris sensory cell modulating agent formulations byreducing the rate of protein aggregation. The anti-aggregation additiveselected depends upon the nature of the conditions to which the aurissensory cell modulating agents, for example auris sensory cellmodulating agent antibodies are exposed. For example, certainformulations undergoing agitation and thermal stress require a differentanti-aggregation additive than a formulation undergoing lyophilizationand reconstitution. Useful anti-aggregation additives include, by way ofexample only, urea, guanidinium chloride, simple amino acids such asglycine or arginine, sugars, polyalcohols, polysorbates, polymers suchas polyethylene glycol and dextrans, alkyl saccharides, such as alkylglycoside, and surfactants.

Other useful formulations optionally include one or moreauris-acceptable antioxidants to enhance chemical stability whererequired. Suitable antioxidants include, by way of example only,ascorbic acid, methionine, sodium thiosulfate and sodium metabisulfite.In one embodiment, antioxidants are selected from metal chelatingagents, thiol containing compounds and other general stabilizing agents.

Still other useful compositions include one or more auris-acceptablesurfactants to enhance physical stability or for other purposes.Suitable nonionic surfactants include, but are not limited to,polyoxyethylene fatty acid glycerides and vegetable oils, e.g.,polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylenealkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

In some embodiments, the auris-acceptable pharmaceutical formulationsdescribed herein are stable with respect to compound degradation over aperiod of any of at least about 1 day, at least about 2 days, at leastabout 3 days, at least about 4 days, at least about 5 days, at leastabout 6 days, at least about 1 week, at least about 2 weeks, at leastabout 3 weeks, at least about 4 weeks, at least about 5 weeks, at leastabout 6 weeks, at least about 7 weeks, at least about 8 weeks, at leastabout 3 months, at least about 4 months, at least about 5 months, or atleast about 6 months. In other embodiments, the formulations describedherein are stable with respect to compound degradation over a period ofat least about 1 week. Also described herein are formulations that arestable with respect to compound degradation over a period of at leastabout 1 month.

In other embodiments, an additional surfactant (co-surfactant) and/orbuffering agent is combined with one or more of the pharmaceuticallyacceptable vehicles previously described herein so that the surfactantand/or buffering agent maintains the product at an optimal pH forstability. Suitable co-surfactants include, but are not limited to: a)natural and synthetic lipophilic agents, e.g., phospholipids,cholesterol, and cholesterol fatty acid esters and derivatives thereof,b) nonionic surfactants, which include for example, polyoxyethylenefatty alcohol esters, sorbitan fatty acid esters (Spans),polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene (20)sorbitan monooleate (Tween 80), polyoxyethylene (20) sorbitanmonostearate (Tween 60), polyoxyethylene (20) sorbitan monolaurate(Tween 20) and other Tweens, sorbitan esters, glycerol esters, e.g.,Myrj and glycerol triacetate (triacetin), polyethylene glycols, cetylalcohol, cetostearyl alcohol, stearyl alcohol, polysorbate 80,poloxamers, poloxamines, polyoxyethylene castor oil derivatives (e.g.,Cremophor® RH40, Cremphor A25, Cremphor A20, Cremophor® EL) and otherCremophors, sulfosuccinates, alkyl sulphates (SLS); PEG glyceryl fattyacid esters such as PEG-8 glyceryl caprylate/caprate (Labrasol), PEG-4glyceryl caprylate/caprate (Labrafac Hydro WL 1219), PEG-32 glyceryllaurate (Gelucire 444/14), PEG-6 glyceryl mono oleate (Labrafil M 1944CS), PEG-6 glyceryl linoleate (Labrafil M 2125 CS); propylene glycolmono- and di-fatty acid esters, such as propylene glycol laurate,propylene glycol caprylate/caprate; Brij® 700, ascorbyl-6-palmitate,stearylamine, sodium lauryl sulfate, polyoxethyleneglyceroltriiricinoleate, and any combinations or mixtures thereof; c) anionicsurfactants include, but are not limited to, calciumcarboxymethylcellulose, sodium carboxymethylcellulose, sodiumsulfosuccinate, dioctyl, sodium alginate, alkyl polyoxyethylenesulfates, sodium lauryl sulfate, triethanolamine stearate, potassiumlaurate, bile salts, and any combinations or mixtures thereof; and d)cationic surfactants such as cetyltrimethylammonium bromide, andlauryldimethylbenzyl-ammonium chloride.

In a further embodiment, when one or more co-surfactants are utilized inthe auris-acceptable formulations of the present disclosure, they arecombined, e.g., with a pharmaceutically acceptable vehicle and ispresent in the final formulation, e.g., in an amount ranging from about0.1% to about 20%, from about 0.5% to about 10%.

In one embodiment, the surfactant has an HLB value of 0 to 20. Inadditional embodiments, the surfactant has an HLB value of 0 to 3, of 4to 6, of 7 to 9, of 8 to 18, of 13 to 15, of 10 to 18.

In one embodiment, diluents are also used to stabilize the auris sensorycell modulating agent or other pharmaceutical compounds because theyprovide a more stable environment. Salts dissolved in buffered solutions(which also can provide pH control or maintenance) are utilized asdiluents, including, but not limited to a phosphate buffered salinesolution. In other embodiments, the gel formulation is isotonic with theendolymph or the perilymph: depending on the portion of the cochlea thatthe auris sensory cell modulating agent formulation is targeted.Isotonic formulations are provided by the addition of a tonicity agent.Suitable tonicity agents include, but are not limited to anypharmaceutically acceptable sugar, salt or any combinations or mixturesthereof, such as, but not limited to dextrose and sodium chloride. Infurther embodiments, the tonicity agents are present in an amount fromabout 100 mOsm/kg to about 500 mOsm/kg. In some embodiments, thetonicity agent is present in an amount from about 200 mOsm/kg to about400 mOsm/kg, from about 280 mOsm/kg to about 320 mOsm/kg. The amount oftonicity agents will depend on the target structure of thepharmaceutical formulation, as described herein.

Useful tonicity compositions also include one or more salts in an amountrequired to bring osmolality of the composition into an acceptable rangefor the perilymph or the endolymph. Such salts include those havingsodium, potassium or ammonium cations and chloride, citrate, ascorbate,borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfiteanions; suitable salts include sodium chloride, potassium chloride,sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In some embodiments, the auris-acceptable gel formulations disclosedherein alternatively or additionally contains preservatives to preventmicrobial growth. Suitable auris-acceptable preservatives for use in theenhanced viscosity formulations described herein include, but are notlimited to benzoic acid, boric acid, p-hydroxybenzoates, alcohols,quarternary compounds, stabilized chlorine dioxide, mercurials, such asmerfen and thiomersal, mixtures of the foregoing and the like.

In a further embodiment, the preservative is, by way of example only, anantimicrobial agent, within the auris-acceptable formulations presentedherein. In one embodiment, the formulation includes a preservative suchas by way of example only, methyl paraben, sodium bisulfite, sodiumthiosulfate, ascorbate, chorobutanol, thimerosal, parabens, benzylalcohol, phenylethanol and others. In another embodiment, the methylparaben is at a concentration of about 0.05% to about 1.0%, about 0.1%to about 0.2%. In a further embodiment, the gel is prepared by mixingwater, methylparaben, hydroxyethylcellulose and sodium citrate. In afurther embodiment, the gel is prepared by mixing water, methylparaben,hydroxyethylcellulose and sodium acetate. In a further embodiment, themixture is sterilized by autoclaving at 120° C. for about 20 minutes,and tested for pH, methylparaben concentration and viscosity beforemixing with the appropriate amount of the auris sensory cell modulatingagent disclosed herein.

Suitable auris-acceptable water soluble preservatives which are employedin the drug delivery vehicle include sodium bisulfite, sodiumthiosulfate, ascorbate, chorobutanol, thimerosal, parabens, benzylalcohol, Butylated hydroxytoluene (BHT), phenylethanol and others. Theseagents are present, generally, in amounts of about 0.001% to about 5% byweight and, preferably, in the amount of about 0.01 to about 2% byweight. In some embodiments, auris-compatible formulations describedherein are free of preservatives.

Round Window Membrane Penetration Enhancers

In another embodiment, the formulation further comprises one or moreround window membrane penetration enhancers. Penetration across theround window membrane is enhanced by the presence of round windowmembrane penetration enhancers. Round window membrane penetrationenhancers are chemical entities that facilitate transport ofcoadministered substances across the round window membrane. Round windowmembrane penetration enhancers are grouped according to chemicalstructure. Surfactants, both ionic and non-ionic, such as sodium laurylsulfate, sodium laurate, polyoxyethylene-20-cetyl ether, laureth-9,sodium dodecylsulfate, dioctyl sodium sulfosuccinate,polyoxyethylene-9-lauryl ether (PLE), Tween® 80,nonylphenoxypolyethylene (NP-POE), polysorbates and the like, functionas round window membrane penetration enhancers. Bile salts (such assodium glycocholate, sodium deoxycholate, sodium taurocholate, sodiumtaurodihydrofusidate, sodium glycodihydrofusidate and the like), fattyacids and derivatives (such as oleic acid, caprylic acid, mono- anddi-glycerides, lauric acids, acylcholines, caprylic acids,acylcarnitines, sodium caprates and the like), chelating agents (such asEDTA, citric acid, salicylates and the like), sulfoxides (such asdimethyl sulfoxide (DMSO), decylmethyl sulfoxide and the like), andalcohols (such as ethanol, isopropanol, glycerol, propanediol and thelike) also function as round window membrane penetration enhancers.

In some embodiments, the auris acceptable penetration enhancer is asurfactant comprising an alkyl-glycoside wherein the alkyl glycoside istetradecyl-β-D-maltoside. In some embodiments, the auris acceptablepenetration enhancer is a surfactant comprising an alkyl-glycosidewherein the alkyl glycoside is dodecyl-maltoside. In certain instances,the penetration enhancing agent is a hyaluronidase. In certaininstances, a hyaluronidase is a human or bovine hyaluronidase. In someinstances, a hyaluronidase is a human hyaluronidase (e.g., hyaluronidasefound in human sperm, PH20 (Halozyme), Hyelenex® (Baxter International,Inc.)). In some instances, a hyaluronidase is a bovine hyaluronidase(e.g., bovine testicular hyaluronidase, Amphadase® (AmphastarPharmaceuticals), Hydase® (PrimaPharm, Inc). In some instances, ahyluronidase is an ovine hyaluronidase, Vitrase® (ISTA Pharmaceuticals).In certain instances, a hyaluronidase described herein is a recombinanthyaluronidase. In some instances, a hyaluronidase described herein is ahumanized recombinant hyaluronidase. In some instances, a hyaluronidasedescribed herein is a pegylated hyaluronidase (e.g., PEGPH20(Halozyme)). In addition, the peptide-like penetration enhancersdescribed in U.S. Pat. Nos. 7,151,191, 6,221,367 and 5,714,167, hereinincorporated by references for such disclosure, are contemplated as anadditional embodiment. These penetration enhancers are amino-acid andpeptide derivatives and enable drug absorption by passive transcellulardiffusion without affecting the integrity of membranes or intercellulartight junctions.

Round Window Membrane Permeable Liposomes

Liposomes or lipid particles may also be employed to encapsulate theauris sensory cell modulating agent formulations or compositions.Phospholipids that are gently dispersed in an aqueous medium formmultilayer vesicles with areas of entrapped aqueous media separating thelipid layers. Sonication, or turbulent agitation, of these multilayervesicles results in the formation of single layer vesicles, commonlyreferred to as liposomes, with sizes of about 10-1000 nm. Theseliposomes have many advantages as auris sensory cell modulating agentsor other pharmaceutical agent carriers. They are biologically inert,biodegradable, non-toxic and non-antigenic. Liposomes are formed invarious sizes and with varying compositions and surface properties.Additionally, they are able to entrap a wide variety of agents andrelease the agent at the site of liposome collapse.

Suitable phospholipids for use in auris-acceptable liposomes here are,for example, phosphatidyl cholines, ethanolamines and serines,sphingomyelins, cardiolipins, plasmalogens, phosphatidic acids andcerebrosides, in particular those which are soluble together with theauris sensory cell modulating agents herein in non-toxic,pharmaceutically acceptable organic solvents. Preferred phospholipidsare, for example, phosphatidyl choline, phosphatidyl ethanolamine,phosphatidyl serine, phosphatidyl inositol, lysophosphatidyl choline,phosphatidyl glycerol and the like, and mixtures thereof especiallylecithin, e.g. soya lecithin. The amount of phospholipid used in thepresent formulation range from about 10 to about 30%, preferably fromabout 15 to about 25% and in particular is about 20%.

Lipophilic additives may be employed advantageously to modifyselectively the characteristics of the liposomes. Examples of suchadditives include by way of example only, stearylamine, phosphatidicacid, tocopherol, cholesterol, cholesterol hemisuccinate and lanolinextracts. The amount of lipophilic additive used range from 0.5 to 8%,preferably from 1.5 to 4% and in particular is about 2%. Generally, theratio of the amount of lipophilic additive to the amount of phospholipidranges from about 1:8 to about 1:12 and in particular is about 1:10.Said phospholipid, lipophilic additive and the auris sensory cellmodulating agent and other pharmaceutical compounds are employed inconjunction with a non-toxic, pharmaceutically acceptable organicsolvent system which dissolve said ingredients. Said solvent system notonly must dissolve the auris sensory cell modulating agent completely,but it also has to allow the formulation of stable single bilayeredliposomes. The solvent system comprises dimethylisosorbide andtetraglycol (glycofurol, tetrahydrofurfuryl alcohol polyethylene glycolether) in an amount of about 8 to about 30%. In said solvent system, theratio of the amount of dimethylisosorbide to the amount of tetraglycolrange from about 2:1 to about 1:3, in particular from about 1:1 to about1:2.5 and preferably is about 1:2. The amount of tetraglycol in thefinal composition thus vary from 5 to 20%, in particular from 5 to 15%and preferably is approximately 10%. The amount of dimethylisosorbide inthe final composition thus range from 3 to 10%, in particular from 3 to7% and preferably is approximately 5%.

The term “organic component” as used hereinafter refers to mixturescomprising said phospholipid, lipophilic additives and organic solvents.The auris sensory cell modulating agent may be dissolved in the organiccomponent, or other means to maintain full activity of the agent. Theamount of auris sensory cell modulating agent in the final formulationmay range from 0.1 to 5.0%. In addition, other ingredients such asanti-oxidants may be added to the organic component. Examples includetocopherol, butylated hydroxyanisole, butylated hydroxytoluene, ascorbylpalmitate, ascorbyl oleate and the like.

Liposomal formulations are alternatively prepared, for auris sensorycell modulating agents or other pharmaceutical agents that aremoderately heat-resistant, by (a) heating the phospholipid and theorganic solvent system to about 60-80° C. in a vessel, dissolving theactive ingredient, then adding any additional formulating agents, andstirring the mixture until complete dissolution is obtained; (b) heatingthe aqueous solution to 90-95° C. in a second vessel and dissolving thepreservatives therein, allowing the mixture to cool and then adding theremainder of the auxiliary formulating agents and the remainder of thewater, and stirring the mixture until complete dissolution is obtained;thus preparing the aqueous component; (c) transferring the organic phasedirectly into the aqueous component, while homogenizing the combinationwith a high performance mixing apparatus, for example, a high-shearmixer; and (d) adding a viscosity enhancing agent to the resultingmixture while further homogenizing. The aqueous component is optionallyplaced in a suitable vessel which is equipped with a homogenizer andhomogenization is effected by creating turbulence during the injectionof the organic component. Any mixing means or homogenizer which exertshigh shear forces on the mixture may be employed. Generally, a mixercapable of speeds from about 1,500 to 20,000 rpm, in particular fromabout 3,000 to about 6,000 rpm may be employed. Suitable viscosityenhancing agents for use in process step (d) are for example, xanthangum, hydroxypropyl cellulose, hydroxypropyl methylcellulose or mixturesthereof. The amount of viscosity enhancing agent depends on the natureand the concentration of the other ingredients and in general rangesfrom about 0.5 to 2.0%, or approximately 1.5%. In order to preventdegradation of the materials used during the preparation of theliposomal formulation, it is advantageous to purge all solutions with aninert gas such as nitrogen or argon, and to conduct all steps under aninert atmosphere. Liposomes prepared by the above described methodusually contain most of the active ingredient bound in the lipid bilayerand separation of the liposomes from unencapsulated material is notrequired.

In other embodiments, the auris-acceptable formulations, including gelformulations and viscosity-enhanced formulations, further includeexcipients, other medicinal or pharmaceutical agents, carriers,adjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts, solubilizers, an antifoaming agent,an antioxidant, a dispersing agent, a wetting agent, a surfactant, andcombinations thereof.

Suitable carriers for use in an auris-acceptable formulation describedherein include, but are not limited to, any pharmaceutically acceptablesolvent compatible with the targeted auris structure's physiologicalenvironment. In other embodiments, the base is a combination of apharmaceutically acceptable surfactant and solvent.

In some embodiments, other excipients include, sodium stearyl fumarate,diethanolamine cetyl sulfate, isostearate, polyethoxylated castor oil,nonoxyl 10, octoxynol 9, sodium lauryl sulfate, sorbitan esters(sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate,sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate,sorbitan tristearate, sorbitan laurate, sorbitan oleate, sorbitanpalmitate, sorbitan stearate, sorbitan dioleate, sorbitansesqui-isostearate, sorbitan sesquistearate, sorbitan tri-isostearate),lecithin pharmaceutical acceptable salts thereof and combinations ormixtures thereof.

In other embodiments, the carrier is a polysorbate. Polysorbates arenonionic surfactants of sorbitan esters. Polysorbates useful in thepresent disclosure include, but are not limited to polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 80 (Tween 80) and anycombinations or mixtures thereof. In further embodiments, polysorbate 80is utilized as the pharmaceutically acceptable carrier.

In one embodiment, water-soluble glycerin-based auris-acceptableenhanced viscosity formulations utilized in the preparation ofpharmaceutical delivery vehicles comprise at least one auris sensorycell modulating agent containing at least about 0.1% of thewater-soluble glycerin compound or more. In some embodiments, thepercentage of auris sensory cell modulating agent is varied betweenabout 1% and about 95%, between about 5% and about 80%, between about10% and about 60% or more of the weight or volume of the totalpharmaceutical formulation. In some embodiments, the amount of thecompound(s) in each therapeutically useful auris sensory cell modulatingagent formulation is prepared in such a way that a suitable dosage willbe obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations are contemplated herein.

If desired, the auris-acceptable pharmaceutical gels also containco-solvents, preservatives, cosolvents, ionic strength and osmolalityadjustors and other excipients in addition to buffering agents. Suitableauris-acceptable water soluble buffering agents are alkali or alkalineearth metal carbonates, phosphates, bicarbonates, citrates, borates,acetates, succinates and the like, such as sodium phosphate, citrate,borate, acetate, bicarbonate, carbonate and tromethamine (TRIS). Theseagents are present in amounts sufficient to maintain the pH of thesystem at 7.4±0.2 and preferably, 7.4. As such, the buffering agent isas much as 5% on a weight basis of the total composition.

Cosolvents are used to enhance auris sensory cell modulating agentsolubility, however, some auris sensory cell modulating agents or otherpharmaceutical compounds are insoluble. These are often suspended in thepolymer vehicle with the aid of suitable suspending or viscosityenhancing agents.

Moreover, some pharmaceutical excipients, diluents or carriers arepotentially ototoxic. For example, benzalkonium chloride, a commonpreservative, is ototoxic and therefore potentially harmful ifintroduced into the vestibular or cochlear structures. In formulating acontrolled release auris sensory cell modulating agent formulation, itis advised to avoid or combine the appropriate excipients, diluents orcarriers to lessen or eliminate potential ototoxic components from theformulation, or to decrease the amount of such excipients, diluents orcarriers. Optionally, a controlled release auris sensory cell modulatingagent formulation includes otoprotective agents, such as antioxidants,alpha lipoic acid, calcium, fosfomycin or iron chelators, to counteractpotential ototoxic effects that may arise from the use of specifictherapeutic agents or excipients, diluents or carriers.

Thof therapeutically acceptable otic formulations:

Example Foramulation Example Characteristics Chitosan tunabledegradation of matrix in vitro glycerophosphate (CGP) tunable TACEinhibitor release in vitro: e.g., ~50% of drug released after 24 hrsbiodegradable compatible with drug delivery to the inner ear suitablefor macromolecules and hydrophobic drugs PEG-PLGA-PEG triblock tunablehigh stability: e.g., maintains mechanical integrity polymers >1 monthin vitro tunable fast release of hydrophilic drugs: e.g., ~50% of drugreleased after 24 hrs, and remainder released over ~5 days tunable slowrelease of hydrophobic drugs: e.g., ~80% released after 8 weeksbiodegradable subcutaneous injection of solution: e.g., gel forms withinseconds and is intact after 1 month PEO-PPO-PEO triblock Tunable sol-geltransition temperature: e.g., decreases copolymers (e.g., withincreasing F127 concentration Pluronic or Poloxameres) (e.g., F127)Chitosan CGP formulation tolerates liposomes: e.g., up to 15 uM/mlglycerophosphate with liposomes. drug-loaded liposomes liposomes tunablyreduce drug release time (e.g., up to 2 weeks in vitro). increase inliposome diameter optionally reduces drug release kinetics (e.g.,liposome size between 100 and 300 nm) release parameters are controlledby changing composition of liposomes

The formulations disclosed herein alternatively encompass anotoprotectant agent in addition to the at least one active agent and/orexcipients, including but not limited to such agents as antioxidants,alpha lipoic acid, calcium, fosfomycin or iron chelators, to counteractpotential ototoxic effects that may arise from the use of specifictherapeutic agents or excipients, diluents or carriers.

Modes of Treatment

Dosing Methods and Schedules

Drugs delivered to the inner ear have been administered systemically viaoral, intravenous or intramuscular routes. However, systemicadministration for pathologies local to the inner ear increases thelikelihood of systemic toxicities and adverse side effects and creates anon-productive distribution of drug in which high levels of drug arefound in the serum and correspondingly lower levels are found at theinner ear.

Intratympanic injection of therapeutic agents is the technique ofinjecting a therapeutic agent behind the tympanic membrane into themiddle and/or inner ear. In one embodiment, the formulations describedherein are administered directly onto the round window membrane viatranstympanic injection. In another embodiment, the auris sensory cellmodulating agent auris-acceptable formulations described herein areadministered onto the round window membrane via a non-transtympanicapproach to the inner ear. In additional embodiments, the formulationdescribed herein is administered onto the round window membrane via asurgical approach to the round window membrane comprising modificationof the crista fenestrae cochleae.

In one embodiment the delivery system is a syringe and needle apparatusthat is capable of piercing the tympanic membrane and directly accessingthe round window membrane or crista fenestrae cochleae of the aurisinterna. In some embodiments, the needle on the syringe is wider than a18 gauge needle. In another embodiment, the needle gauge is from 18gauge to 31 gauge. In a further embodiment, the needle gauge is from 25gauge to 30 gauge. Depending upon the thickness or viscosity of theauris sensory cell modulating agent compositions or formulations, thegauge level of the syringe or hypodermic needle may be variedaccordingly. In another embodiment, the internal diameter of the needlecan be increased by reducing the wall thickness of the needle (commonlyreferred as thin wall or extra thin wall needles) to reduce thepossibility of needle clogging while maintaining an adequate needlegauge.

In another embodiment, the needle is a hypodermic needle used forinstant delivery of the gel formulation. The hypodermic needle may be asingle use needle or a disposable needle. In some embodiments, a syringemay be used for delivery of the pharmaceutically acceptable gel-basedauris sensory cell modulating agent-containing compositions as disclosedherein wherein the syringe has a press-fit (Luer) or twist-on(Luer-lock) fitting. In one embodiment, the syringe is a hypodermicsyringe. In another embodiment, the syringe is made of plastic or glass.In yet another embodiment, the hypodermic syringe is a single usesyringe. In a further embodiment, the glass syringe is capable of beingsterilized. In yet a further embodiment, the sterilization occursthrough an autoclave. In another embodiment, the syringe comprises acylindrical syringe body wherein the gel formulation is stored beforeuse. In other embodiments, the syringe comprises a cylindrical syringebody wherein the auris sensory cell modulating agent pharmaceuticallyacceptable gel-based compositions as disclosed herein is stored beforeuse which conveniently allows for mixing with a suitablepharmaceutically acceptable buffer. In other embodiments, the syringemay contain other excipients, stabilizers, suspending agents, diluentsor a combination thereof to stabilize or otherwise stably store theauris sensory cell modulating agent or other pharmaceutical compoundscontained therein.

In some embodiments, the syringe comprises a cylindrical syringe bodywherein the body is compartmentalized in that each compartment is ableto store at least one component of the auris-acceptable auris sensorycell modulating agent gel formulation. In a further embodiment, thesyringe having a compartmentalized body allows for mixing of thecomponents prior to injection into the auris media or auris interna. Inother embodiments, the delivery system comprises multiple syringes, eachsyringe of the multiple syringes contains at least one component of thegel formulation such that each component is pre-mixed prior to injectionor is mixed subsequent to injection. In a further embodiment, thesyringes disclosed herein comprise at least one reservoir wherein the atleast one reservoir comprises an auris sensory cell modulating agent, ora pharmaceutically acceptable buffer, or a viscosity enhancing agent,such as a gelling agent or a combination thereof. Commercially availableinjection devices are optionally employed in their simplest form asready-to-use plastic syringes with a syringe barrel, needle assemblywith a needle, plunger with a plunger rod, and holding flange, toperform an intratympanic injection.

In some embodiments, the delivery device is an apparatus designed foradministration of therapeutic agents to the middle and/or inner ear. Byway of example only: GYRUS Medical Gmbh offers micro-otoscopes forvisualization of and drug delivery to the round window niche; Arenberghas described a medical treatment device to deliver fluids to inner earstructures in U.S. Pat. Nos. 5,421,818; 5,474,529; and 5,476,446, eachof which is incorporated by reference herein for such disclosure. U.S.patent application Ser. No. 08/874,208, which is incorporated herein byreference for such disclosure, describes a surgical method forimplanting a fluid transfer conduit to deliver therapeutic agents to theinner ear. U.S. Patent Application Publication 2007/0167918, which isincorporated herein by reference for such disclosure, further describesa combined otic aspirator and medication dispenser for intratympanicfluid sampling and medicament application.

The auris-acceptable compositions or formulations containing the aurissensory cell modulating agent compound(s) described herein areadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the auris sensory cell modulating agentcompositions are administered to a patient already suffering from anautoimmune disease, condition or disorder, in an amount sufficient tocure or at least partially arrest the symptoms of the disease, disorderor condition. Amounts effective for this use will depend on the severityand course of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

Frequency of Administration

In some embodiments, a composition disclosed herein is administered toan individual in need thereof once. In some embodiments, a compositiondisclosed herein is administered to an individual in need thereof morethan once. In some embodiments, a first administration of a compositiondisclosed herein is followed by a second administration of a compositiondisclosed herein. In some embodiments, a first administration of acomposition disclosed herein is followed by a second and thirdadministration of a composition disclosed herein. In some embodiments, afirst administration of a composition disclosed herein is followed by asecond, third, and fourth administration of a composition disclosedherein. In some embodiments, a first administration of a compositiondisclosed herein is followed by a second, third, fourth, and fifthadministration of a composition disclosed herein. In some embodiments, afirst administration of a composition disclosed herein is followed by adrug holiday.

The number of times a composition is administered to an individual inneed thereof depends on the discretion of a medical professional, thedisorder, the severity of the disorder, and the individual's response tothe formulation. In some embodiments, a composition disclosed herein isadministered once to an individual in need thereof with a mild acutecondition. In some embodiments, a composition disclosed herein isadministered more than once to an individual in need thereof with amoderate or severe acute condition. In the case wherein the patient'scondition does not improve, upon the doctor's discretion theadministration of an auris sensory cell modulator may be administeredchronically, that is, for an extended period of time, includingthroughout the duration of the patient's life in order to ameliorate orotherwise control or limit the symptoms of the patient's disease orcondition.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the auris sensory cellmodulating agent compounds may be administered chronically, that is, foran extended period of time, including throughout the duration of thepatient's life in order to ameliorate or otherwise control or limit thesymptoms of the patient's disease or condition.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the auris sensory cell modulating agentcompounds may be given continuously; alternatively, the dose of drugbeing administered may be temporarily reduced or temporarily suspendedfor a certain length of time (i.e., a “drug holiday”). The length of thedrug holiday varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, and 365 days. The dose reduction during a drugholiday may be from 10%-100%, including by way of example only 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, and 100%.

Once improvement of the patient's otic conditions has occurred, amaintenance auris sensory cell modulating agent dose is administered ifnecessary. Subsequently, the dosage or the frequency of administration,or both, is optionally reduced, as a function of the symptoms, to alevel at which the improved disease, disorder or condition is retained.In certain embodiments, patients require intermittent treatment on along-term basis upon any recurrence of symptoms.

The amount of auris sensory cell modulating agent that will correspondto such an amount will vary depending upon factors such as theparticular compound, disease condition and its severity, according tothe particular circumstances surrounding the case, including, e.g., thespecific auris sensory cell modulating agent being administered, theroute of administration, the autoimmune condition being treated, thetarget area being treated, and the subject or host being treated. Ingeneral, however, doses employed for adult human treatment willtypically be in the range of 0.02-50 mg per administration, preferably1-15 mg per administration. The desired dose is presented in a singledose or as divided doses administered simultaneously (or over a shortperiod of time) or at appropriate intervals.

In some embodiments, the initial administration is a particular aurissensory cell modulating agent and the subsequent administration adifferent formulation or auris sensory cell modulating agent.

Pharmacokinetics of Controlled Release Formulations

In one embodiment, the formulations disclosed herein additionallyprovides an immediate release of an auris sensory cell modulating agentfrom the composition, or within 1 minute, or within 5 minutes, or within10 minutes, or within 15 minutes, or within 30 minutes, or within 60minutes or within 90 minutes. In other embodiments, a therapeuticallyeffective amount of at least one auris sensory cell modulating agent isreleased from the composition immediately, or within 1 minute, or within5 minutes, or within 10 minutes, or within 15 minutes, or within 30minutes, or within 60 minutes or within 90 minutes. In certainembodiments the composition comprises an auris-pharmaceuticallyacceptable gel formulation providing immediate release of at least oneauris sensory cell modulating agent. Additional embodiments of theformulation may also include an agent that enhances the viscosity of theformulations included herein.

In other or further embodiments, the formulation provides an extendedrelease formulation of at least one auris sensory cell modulating agent.In certain embodiments, diffusion of at least one auris sensory cellmodulating agent from the formulation occurs for a time period exceeding5 minutes, or 15 minutes, or 30 minutes, or 1 hour, or 4 hours, or 6hours, or 12 hours, or 18 hours, or 1 day, or 2 days, or 3 days, or 4days, or 5 days, or 6 days, or 7 days, or 10 days, or 12 days, or 14days, or 18 days, or 21 days, or 25 days, or 30 days, or 45 days, or 2months or 3 months or 4 months or 5 months or 6 months or 9 months or 1year. In other embodiments, a therapeutically effective amount of atleast one auris sensory cell modulating agent is released from theformulation for a time period exceeding 5 minutes, or 15 minutes, or 30minutes, or 1 hour, or 4 hours, or 6 hours, or 12 hours, or 18 hours, or1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days,or 10 days, or 12 days, or 14 days, or 18 days, or 21 days, or 25 days,or 30 days, or 45 days, or 2 months or 3 months or 4 months or 5 monthsor 6 months or 9 months or 1 year.

In other embodiments, the formulation provides both an immediate releaseand an extended release formulation of an auris sensory cell modulatingagent. In yet other embodiments, the formulation contains a 0.25:1ratio, or a 0.5:1 ratio, or a 1:1 ratio, or a 1:2 ratio, or a 1:3, or a1:4 ratio, or a 1:5 ratio, or a 1:7 ratio, or a 1:10 ratio, or a 1:15ratio, or a 1:20 ratio of immediate release and extended releaseformulations. In a further embodiment the formulation provides animmediate release of a first auris sensory cell modulating agent and anextended release of a second auris sensory cell modulating agent orother therapeutic agent. In yet other embodiments, the formulationprovides an immediate release and extended release formulation of atleast one auris sensory cell modulating agent, and at least onetherapeutic agent. In some embodiments, the formulation provides a0.25:1 ratio, or a 0.5:1 ratio, or a 1:1 ratio, or a 1:2 ratio, or a1:3, or a 1:4 ratio, or a 1:5 ratio, or a 1:7 ratio, or a 1:10 ratio, ora 1:15 ratio, or a 1:20 ratio of immediate release and extended releaseformulations of a first auris sensory cell modulating agent and secondtherapeutic agent, respectively.

In a specific embodiment the formulation provides a therapeuticallyeffective amount of at least one auris sensory cell modulating agent atthe site of disease with essentially no systemic exposure. In anadditional embodiment the formulation provides a therapeuticallyeffective amount of at least one auris sensory cell modulating agent atthe site of disease with essentially no detectable systemic exposure. Inother embodiments, the formulation provides a therapeutically effectiveamount of at least one auris sensory cell modulating agent at the siteof disease with little or no detectable systemic exposure.

The combination of immediate release, delayed release and/or extendedrelease auris sensory cell modulating agent compositions or formulationsmay be combined with other pharmaceutical agents, as well as theexcipients, diluents, stabilizers, tonicity agents and other componentsdisclosed herein. As such, depending upon the auris sensory cellmodulating agent used, the thickness or viscosity desired, or the modeof delivery chosen, alternative aspects of the embodiments disclosedherein are combined with the immediate release, delayed release and/orextended release embodiments accordingly.

In certain embodiments, the pharmacokinetics of the auris sensory cellmodulating agent formulations described herein are determined byinjecting the formulation on or near the round window membrane of a testanimal (including by way of example, a guinea pig or a chinchilla). At adetermined period of time (e.g., 6 hours, 12 hours, 1 day, 2 days, 3days, 4 days, 5 days, 6 days, and 7 days for testing thepharmacokinetics of a formulation over a 1 week period), the test animalis euthanized and a 5 mL sample of the perilymph fluid is tested. Theinner ear removed and tested for the presence of the auris sensory cellmodulating agent. As needed, the level of auris sensory cell modulatingagent is measured in other organs. In addition, the systemic level ofthe auris sensory cell modulating agent is measured by withdrawing ablood sample from the test animal. In order to determine whether theformulation impedes hearing, the hearing of the test animal isoptionally tested.

Alternatively, an inner ear is provided (as removed from a test animal)and the migration of the auris sensory cell modulating agent ismeasured. As yet another alternative, an in vitro model of a roundwindow membrane is provided and the migration of the auris sensory cellmodulating agent is measured.

Kits/Articles of Manufacture

The disclosure also provides kits for preventing, treating orameliorating the symptoms of a disease or disorder in a mammal. Suchkits generally will comprise one or more of the auris sensory cellmodulating agent controlled-release compositions or devices disclosedherein, and instructions for using the kit. The disclosure alsocontemplates the use of one or more of the auris sensory cell modulatingagent controlled-release compositions, in the manufacture of medicamentsfor treating, abating, reducing, or ameliorating the symptoms of adisease, dysfunction, or disorder in a mammal, such as a human that has,is suspected of having, or at risk for developing an inner ear disorder.

In some embodiments, kits include a carrier, package, or container thatis compartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) including one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In other embodiments, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arealso presented herein. See, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558and 5,033,252. Examples of pharmaceutical packaging materials include,but are not limited to, blister packs, bottles, tubes, inhalers, pumps,bags, vials, containers, syringes, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment. A wide array of auris sensory cell modulating agentformulations compositions provided herein are contemplated as are avariety of treatments for any disease, disorder, or condition that wouldbenefit by controlled release administration of an auris sensory cellmodulating agent to the inner ear.

In some embodiments, a kit includes one or more additional containers,each with one or more of various materials (such as reagents, optionallyin concentrated form, and/or devices) desirable from a commercial anduser standpoint for use of a formulation described herein. Non-limitingexamples of such materials include, but not limited to, buffers,diluents, filters, needles, syringes; carrier, package, container, vialand/or tube labels listing contents and/or instructions for use andpackage inserts with instructions for use. A set of instructions isoptionally included. In a further embodiment, a label is on orassociated with the container. In yet a further embodiment, a label ison a container when letters, numbers or other characters forming thelabel are attached, molded or etched into the container itself; a labelis associated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Inother embodiments a label is used to indicate that the contents are tobe used for a specific therapeutic application. In yet anotherembodiment, a label also indicates directions for use of the contents,such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. In another embodiment, the packfor example contains metal or plastic foil, such as a blister pack. In afurther embodiment, the pack or dispenser device is accompanied byinstructions for administration. In yet a further embodiment, the packor dispenser is also accompanied with a notice associated with thecontainer in form prescribed by a governmental agency regulating themanufacture, use, or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the drug for human orveterinary administration. In another embodiment, such notice, forexample, is the labeling approved by the U.S. Food and DrugAdministration for prescription drugs, or the approved product insert.In yet another embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES Example 1 Preparation of a Thermoreversible Gel AMN082Formulation

Quantity (mg/g of Ingredient formulation) AMN082 3.0 Methylparaben 0.3Hypromellose 3.0 Poloxamer 407 54 TRIS HCl buffer (0.1 M) 239.7

AMN082 is supplied as a solid. It is rehydrated in water to a finalmolarity of 10 mM.

A 10-g batch of gel formulation containing 1.0% of AMN082 is prepared byfirst suspending Poloxamer 407 (BASF Corp.) in TRIS HCl buffer (0.1 M).The Poloxamer 407 and TRIS are mixed under agitation overnight at 4° C.to ensure complete dissolution of the Poloxamer 407 in the TRIS. Thehypromellose, methylparaben and additional TRIS HCl buffer (0.1 M) isadded. The composition is stirred until dissolution is observed. Asolution of AMN082 is added and the composition is mixed until ahomogenous gel is produced. The mixture is maintained below roomtemperature until use.

Example 2 Preparation of a Mucoadhesive, Thermoreversible Gel AMN082Formulation

Quantity (mg/g of Ingredient formulation) AMN082 3.0 methylparaben 0.3Hypromellose 3.0 Carbopol 934P 0.6 Poloxamer 407 54 TRIS HCl buffer (0.1M) 239.1

AMN082 is supplied as a solid. It is rehydrated in water to a finalmolarity of 10 mM.

A 10-g batch of mucoadhesive gel formulation containing 1.0% of AMN082is prepared by first suspending Poloxamer 407 (BASF Corp.) and Carbopol934P in TRIS HCl buffer (0.1 M). The Poloxamer 407, Carbopol 934P andTRIS are mixed under agitation overnight at 4° C. to ensure completedissolution of the Poloxamer 407 and Carbopol 934P in the TRIS. Thehypromellose, methylparaben and additional TRIS HCl buffer (0.1 M) isadded. The composition is stirred until dissolution is observed. TheAMN082 solution is added and the composition is mixed until a homogenousgel is produced. The mixture is maintained below room temperature untiluse.

Example 3 Preparation of a Hydrogel-Based CNQX Formulation

Quantity (mg/g of Ingredient formulation) CNQX 15.0 paraffin oil 300.0 trihydroxystearate 15.0 cetyl dimethicon copolyol 45.0 water qs ad 1000phosphate buffer pH 7.4 qs pH 7.4

The cream-type formulation is first prepared by gently mixing CNQX withwater until the CNQX is dissolved. Then, the oil base is prepared bymixing paraffin oil, trihydroxystearate and cetyl dimethicon copolyol attemperatures up to 60° C. The oil base is cooled to room temperature andthe CNQX solution is added. The two phases are mixed until a homogenous,monophasic hydrogel is formed.

Example 4 Preparation of a Gel Carbamazepine Formulation

Quantity (mg/g of Ingredient formulation) Carbamazepine 6.4 chitosan 3.2Glycerophosphate disodium 12.8 water 134.4

A 5 ml solution of acetic acid is titrated to a pH of about 4.0. Thechitosan is added to achieve a pH of about 5.5. The carbamazepine isthen dissolved in the chitosan solution. This solution is sterilized byfiltration. A 5 ml aqueous solution of glycerophosphate disodium is alsoprepared and sterilized. The two solutions are mixed and within 2 h at37° C., the desired gel is formed.

Example 5 Preparation of a Mucoadhesive, Thermoreversible GelD-Methionine Formulation

Quantity (mg/g of Ingredient formulation) D-Methionine 3.0 Benzylalcohol 0.3 Hypromellose 3.0 Carbopol 934P 0.6 Poloxamer 407 54  Phosphate buffer, pH 7.4 qs to grams

D-Methionine is dissolved in phosphate buffer. A 10-g batch ofmucoadhesive gel formulation containing D-methionine is prepared byfirst suspending Poloxamer 407 (BASF Corp.) and Carbopol 934P inphosphate buffer. The Poloxamer 407, Carbopol 934P and phosphate bufferare mixed under agitation overnight at 4° C. to ensure completedissolution of the Poloxamer 407 and Carbopol 934P in the buffer. Thehypromellose, benzyl alcohol and additional phosphate buffer is added tothe mixture. The composition is stirred until dissolution is observed.The D-methionine solution is added and the composition is mixed until ahomogenous gel is produced. The mixture is maintained below roomtemperature until use.

Example 6 Preparation of a Liposomal AMN082 Formulation

Ingredient Quantity (mg/g) AMN082 3.0 soya lecithin 200.0 cholesterol20.0 tetraglycol 100.0 dimethylisosorbide 50.0 methylparaben 2.0propylparaben 0.2 BHT 0.1 sodium chloride 1.0 HPMC 15.0 sodium hydroxide0.6 citric acid 1.0 purified water, USP 603.6

AMN082 is supplied as a solid. It is rehydrated in water to a finalmolarity of 10 mM.

Heat the soya lecithin, tetraglycol and dimethyl isosorbide to about70-75° C. Dissolve the cholesterol and butylated hydroxytoluene in theheated mixture. Stir until complete dissolution is obtained. Heat aboutone third of the water to 80-95° C. in a separate vessel and dissolvethe preservatives methylparaben and propylparaben in the heated waterwhile stirring. Allow the solution to cool to about 25° C. and then addthe AMN082, disodium edetate, sodium chloride, sodium hydroxide andcitric acid. Add the remainder of the water and stir to obtain acomplete solution. Transfer the organic mixture into the aqueous mixtureby means of a vacuum, while homogenizing the combination with ahigh-shear mixer until a homogeneous product is obtained. Add thehydroxypropyl methylcellulose into the biphasic mixture by means of avacuum while homogenizing with a mixer. Single bilayered liposomes areformed.

Example 7 Preparation of a Thermoreversible Gel Comprising (S)-Ketamine

Quantity (mg/g of Ingredient formulation) (S)-ketamine 21.0methylparaben 2.1 Hypromellose 21.0 Poloxamer 407 378 TRIS HCl buffer(0.1 M) 1677.9

A 10-g batch of gel formulation containing 1.0% of (S)-ketamine isprepared by first suspending Poloxamer 407 (BASF Corp.) in TRIS HClbuffer (0.1 M). The Poloxamer 407 and TRIS are mixed under agitationovernight at 4° C. to ensure complete dissolution of the Poloxamer 407in the TRIS. The hypromellose, methylparaben and additional TRIS HClbuffer (0.1 M) is added. The composition is stirred until dissolution isobserved. A solution of (S)-ketamine is added and the composition ismixed until a homogenous gel is produced. The mixture is maintainedbelow room temperature until use.

Example 8 Preparation of a Thermoreversible Gel (S)-Ketamine CompositionComprising Micronized (S)-Ketamine Powder

Quantity (mg/g of Ingredient formulation) (S)-Ketamine 20.0 BHT 0.002Poloxamer 407 160.0 PBS buffer (0.1 M) 9.0

A 10-g batch of gel formulation containing 2.0% of micronized(S)-Ketamine, 13.8 mg of sodium phosphate dibasic dihydrate USP (FisherScientific.)+3.1 mg of sodium phosphate monobasic monohydrate USP(Fisher Scientific.)+74 mg of sodium chloride USP (Fisher Scientific.)is dissolved with 8.2 g of sterile filtered DI water and the pH isadjusted to 7.4 with 1 M NaOH. The buffer solution is chilled down and1.6 g of poloxamer 407 (BASF Corp., containing approximately 100 ppm ofBHT) is sprinkled into the chilled PBS solution while mixing, solutionis mixed until all the poloxamer is dissolved. The poloxamer is sterilefiltered using a 33 mm PVDF 0.22 μm sterile syringe filter (MilliporeCorp.) and delivered to 2 mL sterile glass vials (Wheaton) in an asepticenvironment, the vials are closed with sterile butyl rubber stoppers(Kimble) and crimped sealed with 13 mm Al seals (Kimble). 20 mg ofmicronized (S)-ketamine is placed in separate clean depyrogenated vials,the vials are closed with sterile butyl rubber stoppers (Kimble) andcrimped sealed with 13 mm Al seals (Kimble), vials are dry heatsterilized (Fisher Scientific Isotemp oven) for 7 hours at 140° C.Before administration for the experiments described herein, 1 mL of thecold poloxamer solution is delivered to a vial containing 20 mg ofsterile micronized (S)-ketamine using a 21G needle (Becton Dickinson)attached to a 1 mL sterile syringe (Becton Dickinson), suspension mixedwell by shaking to ensure homogeneity of the suspension. The suspensionis then withdrawn with the 21G syringe and the needle is switched to a27 G needle for administration.

Example 9 Preparation of a Thermoreversible Gel Micronized AM-101Composition Comprising a Penetration Enhancer

Quantity (mg/g of Ingredient formulation) AM-101 20.0 methylparaben 1.0Dodecyl maltoside (A3) 1.0 HPMC 10.0 Poloxamer 407 180.0 TRIS HCl buffer(0.1 M) 789.0

A 10-g batch of gel formulation containing 2.0% of micronized AM-101 isprepared by suspending 1.80 g of Poloxamer 407 (BASF Corp.) in 5.00 g ofTRIS HCl buffer (0.1 M) and the components are mixed under agitationovernight at 4° C. to ensure complete dissolution. (S)-Ketamine (200.0mg), hydroxypropylmethylcellulose (100.0 mg), methylparaben (10 mg) anddodecyl maltoside (10 mg) and additional TRIS HCl buffer (0.1 M) (2.89g) is added and further stirring allowed until complete dissolution isobserved. The mixture is maintained below room temperature until use.

Example 10 Effect of pH on Degradation Products for Autoclaved 17%Poloxamer 407NF/2% Otic Agent in PBS Buffer

A stock solution of a 17% poloxamer 407/2% otic agent is prepared bydissolving 351.4 mg of sodium chloride (Fisher Scientific), 302.1 mg ofsodium phosphate dibasic anhydrous (Fisher Scientific), 122.1 mg ofsodium phosphate monobasic anhydrous (Fisher Scientific) and anappropriate amount of an otic agent with 79.3 g of sterile filtered DIwater. The solution is cooled down in a ice chilled water bath and then17.05 g of poloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into thecold solution while mixing. The mixture is further mixed until thepoloxamer is completely dissolved. The pH for this solution is measured.

17% poloxamer 407/2% otic agent in PBS pH of 5.3. Take an aliquot(approximately 30 mL) of the above solution and adjust the pH to 5.3 bythe addition of 1 M HCl.

17% poloxamer 407/2% otic agent in PBS pH of 8.0. Take an aliquot(approximately 30 mL) of the above stock solution and adjust the pH to8.0 by the addition of 1 M NaOH.

A PBS buffer (pH 7.3) is prepared by dissolving 805.5 mg of sodiumchloride (Fisher Scientific), 606 mg of sodium phosphate dibasicanhydrous (Fisher Scientific), 247 mg of sodium phosphate monobasicanhydrous (Fisher Scientific), then QS to 200 g with sterile filtered DIwater.

A 2% solution of an otic agent in PBS pH 7.3 is prepared by dissolvingan appropriate amount of the otic agent in the PBS buffer and QS to 10 gwith PBS buffer.

One mL samples are individually placed in 3 mL screw cap glass vials(with rubber lining) and closed tightly. The vials are placed in aMarket Forge-sterilmatic autoclave (settings, slow liquids) andsterilized at 250° F. for 15 minutes. After the autoclave the samplesare left to cool down to room temperature and then placed inrefrigerator. The samples are homogenized by mixing the vials whilecold.

Appearance (e.g., discoloration and/or precipitation) is observed andrecorded. HPLC analysis is performed using an Agilent 1200 equipped witha Luna C18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrilegradient (1-10 min) of (water-acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 30 μLof sample and dissolved with 1.5 mL of a 1:1 acetonitrile water mixture.Purity of the otic agent in the autoclaved samples is recorded.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, prepared according to the procedure above, aretested using the above procedure to determine the effect of pH ondegradation during the autoclaving step.

Example 11 Effect of Autoclaving on the Release Profile and Viscosity ofa 17% Poloxamer 407NF/2% Otic Agent in PBS

An aliquot of a sample (autoclaved and not autoclaved) is evaluated forrelease profile and viscosity measurement to evaluate the impact of heatsterilization on the properties of the gel.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm). 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL is placed intoreservoir and shaken using a Labline orbit shaker at 70 rpm. Samples aretaken every hour (0.1 mL withdrawn and replace with warm buffer).Samples are analyzed for poloxamer concentration by UV at 624 nm usingthe cobalt thiocyanate method, against an external calibration standardcurve. In brief, 20 μL of the sample is mixed with 1980 μL of a 15 mMcobalt thiocyanate solution and absorbance measured at 625 nm, using aEvolution 160 UV/Vis spectrophotometer (Thermo Scientific).

The released otic agent is fitted to the Korsmeyer-Peppas equation

$\frac{Q}{Q_{\alpha}} = {{kt}^{n} + b}$

where Q is the amount of otic agent released at time t, Q_(a) is theoverall released amount of otic agent, k is a release constant of thenth order, n is a dimensionless number related to the dissolutionmechanism and b is the axis intercept, characterizing the initial burstrelease mechanism wherein n=1 characterizes an erosion controlledmechanism. The mean dissolution time (MDT) is the sum of differentperiods of time the drug molecules stay in the matrix before release,divided by the total number of molecules and is calculated by:

${MDT} = \frac{{nk}^{{- 1}/n}}{n + 1}$

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, prepared according to the procedures describedabove, are tested using the procedure described above to determine Tgel.

Example 12 Effect of Addition of a Secondary Polymer on the DegradationProducts and Viscosity of a Formulation Containing 2% Otic Agent and 17%Poloxamer 407NF after Heat Sterilization (Autoclaving)

Solution A. A solution of pH 7.0 comprising sodiumcarboxymethylcellulose (CMC) in PBS buffer is prepared by dissolving178.35 mg of sodium chloride (Fisher Scientific), 300.5 mg of sodiumphosphate dibasic anhydrous (Fisher Scientific), 126.6 mg of sodiumphosphate monobasic anhydrous (Fisher Scientific) dissolved with 78.4 ofsterile filtered DI water, then 1 g of Blanose 7M65 CMC (Hercules,viscosity of 5450 cP @ 2%) is sprinkled into the buffer solution andheated to aid dissolution, and the solution is then cooled down.

A solution of pH 7.0 comprising 17% poloxamer 407NF/1% CMC/2% otic agentin PBS buffer is made by cooling down 8.1 g of solution A in a icechilled water bath and then adding an appropriate amount of an oticagent followed by mixing. 1.74 g of poloxamer 407NF (Spectrum Chemicals)is sprinkled into the cold solution while mixing. The mixture is furthermixed until all the poloxamer is completely dissolved.

Two mL of the above sample is placed in a 3 mL screw cap glass vial(with rubber lining) and closed tightly. The vial is placed in a MarketForge-sterilmatic autoclave (settings, slow liquids) and sterilized at250° F. for 25 minutes. After autoclaving the sample is left to cooldown to room temperature and then placed in refrigerator. The sample ishomogenized by mixing while the vials are cold.

Precipitation or discoloration are observed after autoclaving. HPLCanalysis is performed using an Agilent 1200 equipped with a Luna C18(2)3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrile gradient(1-10 min) of (water-acetonitrile mixture containing 0.05% TFA), for atotal run of 15 minutes. Samples are diluted by taking 30 μL of sampleand dissolving with 1.5 mL of a 1:1 acetonitrile water mixture. Purityof the otic agent in the autoclaved samples is recorded.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Dissolution is performed at 37° C. for the non-autoclaved sample insnapwells (6.5 mm diameter polycarbonate membrane with a pore size of0.4 μm), 0.2 mL of gel is placed into snapwell and left to harden, then0.5 mL is placed into reservoir and shaken using a Labline orbit shakerat 70 rpm. Samples are taken every hour (0.1 mL withdrawn and replacedwith warm buffer). Samples are analyzed for otic agent concentration byUV at 245 nm, against an external calibration standard curve.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, are tested using the above procedure todetermine the effect addition of a secondary polymer on the degradationproducts and viscosity of a formulation containing 2% otic agent and 17%poloxamer 407NF after heat sterilization (autoclaving).

Example 13 Effect of Buffer Type on the Degradation Products forFormulations Containing Poloxamer 407NF after Heat Sterilization(Autoclaving)

A TRIS buffer is made by dissolving 377.8 mg of sodium chloride (FisherScientific), and 602.9 mg of Tromethamine (Sigma Chemical Co.) then QSto 100 g with sterile filtered DI water, pH is adjusted to 7.4 with 1MHCl.

Stock Solution Containing 25% Poloxamer 407 Solution in TRIS Buffer:

Weigh 45 g of TRIS buffer, chill in an ice chilled bath then sprinkleinto the buffer, while mixing, 15 g of poloxamer 407 NF (SpectrumChemicals). The mixture is further mixed until all the poloxamer iscompletely dissolved.

A series of formulations is prepared with the above stock solution. Anappropriate amount of otic agent (or salt or prodrug thereof) and/orotic agent as micronized/coated/liposomal particles (or salt or prodrugthereof) is used for all experiments.

Stock Solution (pH 7.3) Containing 25% Poloxamer 407 Solution in PBSBuffer:

PBS buffer described above is used. Dissolve 704 mg of sodium chloride(Fisher Scientific), 601.2 mg of sodium phosphate dibasic anhydrous(Fisher Scientific), 242.7 mg of sodium phosphate monobasic anhydrous(Fisher Scientific) with 140.4 g of sterile filtered DI water. Thesolution is cooled down in an ice chilled water bath and then 50 g ofpoloxamer 407NF (SPECTRUM CHEMICALS) is sprinkled into the cold solutionwhile mixing. The mixture is further mixed until the poloxamer iscompletely dissolved.

A series of formulations is prepared with the above stock solution. Anappropriate amount of otic agent (or salt or prodrug thereof) and/orotic agent as micronized/coated/liposomal particles (or salt or prodrugthereof) is used for all experiments.

Tables 2 and 3 list samples prepared using the procedures describedabove. An appropriate amount of otic agent is added to each sample toprovide a final concentration of 2% otic agent in the sample.

TABLE 2 Preparation of samples containing TRIS buffer 25% Stock SolutionTRIS Buffer Sample pH (g) (g) 20% P407/2% otic agent/TRIS 7.45 8.01 1.8218% P407/2% otic agent/TRIS 7.45 7.22 2.61 16% P407/2% otic agent/TRIS7.45 6.47 3.42 18% P407/2% otic agent/TRIS 7.4 7.18 2.64  4% oticagent/TRIS 7.5 — 9.7  2% otic agent/TRIS 7.43 — 5  1% otic agent/TRIS7.35 — 5  2% otic agent/TRIS 7.4 — 4.9 (suspension)

TABLE 3 Preparation of samples containing PBS buffer (pH of 7.3) 25%Stock Solution Sample in PBS (g) PBS Buffer (g) 20% P407/2% otic agent/8.03 1.82 PBS 18% P407/2% otic agent/ 7.1 2.63 PBS 16% P407/2% oticagent/ 6.45 3.44 PBS 18% P407/2% otic agent/ — 2.63 PBS  2% oticagent/PBS — 4.9

One mL samples are individually placed in 3 mL screw cap glass vials(with rubber lining) and closed tightly. The vials are placed in aMarket Forge-sterilmatic autoclave (setting, slow liquids) andsterilized at 250° F. for 25 minutes. After the autoclaving the samplesare left to cool down to room temperature. The vials are placed in therefrigerator and mixed while cold to homogenize the samples.

HPLC analysis is performed using an Agilent 1200 equipped with a LunaC18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-80 acetonitrilegradient (1-10 min) of (water-acetonitrile mixture containing 0.05%TFA), for a total run of 15 minutes. Samples are diluted by taking 30 μLof sample and dissolving with 1.5 mL of a 1:1 acetonitrile watermixture. Purity of the otic agent in the autoclaved samples is recorded.The stability of formulations in TRIS and PBS buffers is compared.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition. Only formulations that show no changeafter autoclaving are analyzed.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, are tested using the above procedure todetermine the effect addition of a secondary polymer on the degradationproducts and viscosity of a formulation containing 2% otic agent and 17%poloxamer 407NF after heat sterilization (autoclaving). Stability offormulations containing micronized otic agent is compared tonon-micronized otic agent formulation counterparts.

Example 14 Pulsed Release Otic Formulations

A combination of D-methionine and D-methionine hydrochloride (ratio of1:1) is used to prepare a pulsed release otic agent formulation usingthe procedures described herein. 20% of the delivered dose of D-methioneis solubilized in a 17% poloxamer solution of example 10 with the aid ofbeta-cyclodextrins. The remaining 80% of the otic agent is then added tothe mixture and the final formulation is prepared using any proceduredescribed herein.

Pulsed release formulations comprising DNQX, D-methionine, micronizedAM-101 or micronized (S)-ketamine, prepared according to the proceduresand examples described above, are tested using procedures describedherein to determine pulse release profiles.

Example 15 Preparation of a 17% Poloxamer 407/2% Otic Agent/78 ppm EvansBlue in PBS

A Stock solution of Evans Blue (5.9 mg/mL) in PBS buffer is prepared bydissolving 5.9 mg of Evans Blue (Sigma Chemical Co) with 1 mL of PBSbuffer (from example 10).

A Stock solution containing 25% Poloxamer 407 solution in PBS buffer isused in this study. An appropriate amount of an otic agent is added tothe stock solution to prepare formulations comprising 2% of an oticagent (Table 4).

TABLE 4 Preparation of poloxamer 407 samples containing Evans Blue 25%P407 in Evans Blue Sample ID PBS (g) PBS Buffer (g) Solution (μL) 17%P407/2% otic agent/ 13.6 6 265 EB 20% P407/2% otic agent/ 16.019 3.62265 EB 25% P407/2% otic agent/ 19.63 — 265 EB

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, are prepared according to the proceduresdescribed above and are sterile filtered through 0.22 μm PVDF syringefilters (Millipore corporation), and autoclaved.

The above formulations are dosed to guinea pigs in the middle ear byprocedures described herein and the ability of formulations to gel uponcontact and the location of the gel is identified after dosing and at 24hours after dosing.

Example 16 Terminal Sterilization of Poloxamer 407 Formulations with andwithout a Visualization Dye

17% poloxamer 407/2% otic agent/in phosphate buffer, pH 7.3: Dissolve709 mg of sodium chloride (Fisher Scientific), 742 mg of sodiumphosphate dibasic dehydrate USP (Fisher Scientific), 251.1 mg of sodiumphosphate monobasic monohydrate USP (Fisher Scientific) and anappropriate amount of an otic agent with 158.1 g of sterile filtered DIwater. The solution is cooled down in an ice chilled water bath and then34.13 g of poloxamer 407NF (Spectrum chemicals) is sprinkled into thecold solution while mixing. The mixture is further mixed until thepoloxamer is completely dissolved.

17% poloxamer 407/2% otic agent/59 ppm Evans blue in phosphate buffer:Take two mL of the 17% poloxamer 407/2% otic agent/in phosphate buffersolution and add 2 mL of a 5.9 mg/mL Evans blue (Sigma-Aldrich chemicalCo) solution in PBS buffer.

25% poloxamer 407/2% otic agent/in phosphate buffer: Dissolve 330.5 mgof sodium chloride (Fisher Scientific), 334.5 mg of sodium phosphatedibasic dehydrate USP (Fisher Scientific), 125.9 mg of sodium phosphatemonobasic monohydrate USP (Fisher Scientific) and an appropriate amountof an otic agent with 70.5 g of sterile filtered DI water.

The solution is cooled down in an ice chilled water bath and then 25.1 gof poloxamer 407NF (Spectrum chemicals) is sprinkled into the coldsolution while mixing. The mixture is further mixed until the poloxameris completely dissolved.

25% poloxamer 407/2% otic agent/59 ppm Evans blue in phosphate buffer:Take two mL of the 25% poloxamer 407/2% otic agent/in phosphate buffersolution and add 2 mL of a 5.9 mg/mL Evans blue (Sigma-Aldrich chemicalCo) solution in PBS buffer.

Place 2 mL of formulation into a 2 mL glass vial (Wheaton serum glassvial) and seal with 13 mm butyl str (kimble stoppers) and crimp with a13 mm aluminum seal. The vials are placed in a Market Forge-sterilmaticautoclave (settings, slow liquids) and sterilized at 250° F. for 25minutes. After the autoclaving the samples are left to cool down to roomtemperature and then placed in refrigeration. The vials are placed inthe refrigerator and mixed while cold to homogenize the samples. Samplediscoloration or precipitation after autoclaving is recorded.

HPLC analysis is performed using an Agilent 1200 equipped with a LunaC18(2) 3 μm, 100 Å, 250×4.6 mm column) using a 30-95 methanol:acetatebuffer pH 4 gradient (1-6 min), then isocratic for 11 minutes, for atotal run of 22 minutes. Samples are diluted by taking 30 μL of sampleand dissolved with 0.97 mL of water. The main peaks are recorded in thetable below. Purity before autoclaving is always greater than 99% usingthis method.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-51 spindle rotated at 0.08 rpm (shear rate of 0.31s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 15-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, prepared according to the procedures describedherein, are tested using the above procedures to determine stability ofthe formulations.

Example 17 In Vitro Comparison of Release Profile

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm), 0.2 mL of a gelformulation described herein is placed into snapwell and left to harden,then 0.5 mL buffer is placed into reservoir and shaken using a Lablineorbit shaker at 70 rpm. Samples are taken every hour (0.1 mL withdrawnand replace with warm buffer). Samples are analyzed for otic agentconcentration by UV at 245 nm against an external calibration standardcurve. Pluronic concentration is analyzed at 624 nm using the cobaltthiocyanate method. Relative rank-order of mean dissolution time (MDT)as a function of % P407 is determined. A linear relationship between theformulations mean dissolution time (MDT) and the P407 concentrationindicates that the otic agent is released due to the erosion of thepolymer gel (poloxamer) and not via diffusion. A non-linear relationshipindicates release of otic agent via a combination of diffusion and/orpolymer gel degradation.

Alternatively, samples are analyzed using the method described by LiXin-Yu paper [Acta Pharmaceutica Sinica 2008, 43(2):208-203] andRank-order of mean dissolution time (MDT) as a function of % P407 isdetermined.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, prepared according to the procedures describedherein, are tested using the above procedure to determine the releaseprofile of the otic agents.

Example 18 In Vitro Comparison of Gelation Temperature

The effect of Poloxamer 188 and an otic agent on the gelationtemperature and viscosity of Poloxamer 407 formulations is evaluatedwith the purpose of manipulating the gelation temperature.

A 25% Poloxamer 407 stock solution in PBS buffer and the PBS solutiondescribed above are used. Poloxamer 188NF from BASF is used. Anappropriate amount of otic agent is added to the solutions described inTable 5 to provide a 2% formulation of the otic agent.

TABLE 5 Preparation of samples containing poloxamer 407/poloxamer 18825% P407 Stock Poloxamer PBS Buffer Sample Solution (g) 188 (mg) (g) 16%P407/10% P188 3.207 501 1.3036 17% P407/10% P188 3.4089 500 1.1056 18%P407/10% P188 3.6156 502 0.9072 19% P407/10% P188 3.8183 500 0.7050 20%P407/10% P188 4.008 501 0.5032 20% P407/5% P188 4.01 256 0.770

Mean dissolution time, viscosity and gel temperature of the aboveformulations are measured using procedures described herein.

An equation is fitted to the data obtained and can be utilized toestimate the gelation temperature of F127/F68 mixtures (for 17-20% F127and 0-10% F68).

T _(gel)=−1.8(%F127)+1.3(%F68)+53

An equation is fitted to the data obtained and can be utilized toestimate the Mean Dissolution Time (hr) based on the gelationtemperature of F127/F68 mixtures (for 17-25% F127 and 0-10% F68), usingresults obtained in examples above.

MDT=−0.2(T _(gel))+8

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, are prepared by addition of an appropriateamount of otic agents to the solutions described in Table 5. The geltemperature of the formulations is determined using the proceduredescribed above.

Example 19 Determination of Temperature Range for Sterile Filtration

The viscosity at low temperatures is measured to help guide thetemperature range at which the sterile filtration needs to occur toreduce the possibility of clogging.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-40 spindle rotated at 1, 5 and 10 rpm (shear rateof 7.5, 37.5 and 75 s⁻¹), equipped with a water jacketed temperaturecontrol unit (temperature ramped from 10-25° C. at 1.6° C./min).

The Tgel of a 17% Pluronic P407 is determined as a function ofincreasing concentration of otic agent. The increase in Tgel for a 17%pluronic formulation is estimated by:

ΔT_(gel)=0.93[% otic agent]

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, prepared according to procedures describedherein, are tested using the above procedure to determine thetemperature range for sterile filtration. The effect of addition ofincreased amounts of otic agent on the Tgel, and the apparent viscosityof the formulations is recorded.

Example 20 Determination of Manufacturing Conditions

TABLE 6 Viscosity of potential formulations at manufacturing/filtrationconditions. Apparent Viscosity^(a) (cP) Temperature @ Sample 5° C. belowTgel 20° C. 100 cP Placebo 52 cP @ 17° C. 120 cP   19° C. 17% P407/2%otic 90 cP @ 18° C. 147 cP 18.5° C. agent 17% P407/6% otic 142 cP @ 22°C.  105 cP 19.7° C. agent ^(a)Viscosity measured at a shear rate of 37.5s⁻¹

An 8 liter batch of a 17% P407 placebo is manufactured to evaluate themanufacturing/filtration conditions. The placebo is manufactured byplacing 6.4 liters of DI water in a 3 gallon SS pressure vessel, andleft to cool down in the refrigerator overnight. The following morningthe tank is taken out (water temperature 5° C., RT 18° C.) and 48 g ofsodium chloride, 29.6 g of sodium phosphate dibasic dehydrate and 10 gof sodium phosphate monobasic monohydrate is added and dissolved with anoverhead mixer (IKA RW20 @ 1720 rpm). Half hour later, once the bufferis dissolved (solution temperature 8° C., RT 18° C.), 1.36 kg ofpoloxamer 407 NF (spectrum chemicals) is slowly sprinkled into thebuffer solution in a 15 minute interval (solution temperature 12° C., RT18° C.), then speed is increased to 2430 rpm. After an additional onehour mixing, mixing speed is reduced to 1062 rpm (complete dissolution).

The temperature of the room is maintained below 25° C. to retain thetemperature of the solution at below 19° C. The temperature of thesolution is maintained at below 19° C. up to 3 hours of the initiationof the manufacturing, without the need to chill/cool the container.

Three different Sartoscale (Sartorius Stedim) filters with a surfacearea of 17.3 cm² are evaluated at 20 psi and 14° C. of solution

1) Sartopore 2, 0.2 μm 5445307HS-FF (PES), flow rate of 16 mL/min

2) Sartobran P, 0.2 μm 5235307HS-FF (cellulose ester), flow rate of 12mL/min

3) Sartopore 2 XLI, 0.2 μm 5445307IS-FF (PES), flow rate of 15 mL/min

Sartopore 2 filter 5441307H4-SS is used, filtration is carried out atthe solution temperature using a 0.45, 0.2 μm Sartopore 2 150 sterilecapsule (Sartorius Stedim) with a surface area of 0.015 m² at a pressureof 16 psi. Flow rate is measured at approximately 100 mL/min at 16 psi,with no change in flow rate while the temperature is maintained in the6.5-14° C. range. Decreasing pressure and increasing temperature of thesolution causes a decrease in flow rate due to an increase in theviscosity of the solution. Discoloration of the solution is monitoredduring the process.

TABLE 7 Predicted filtration time for a 17% poloxamer 407 placebo at asolution temperature range of 6.5-14° C. using Sartopore 2, 0.2 μmfilters at a pressure of 16 psi of pressure. Estimated flow rate Time tofilter 8 L Filter Size (m²) (mL/min) (estimated) Sartopore 2, size 40.015 100 mL/min 80 min Sartopore 2, size 7 0.05 330 mL/min 24 minSartopore 2, size 8 0.1 670 mL/min 12 min

Viscosity, Tgel and UV/Vis absorption is check before filtrationevaluation. Pluronic UV/Vis spectra are obtained by a Evolution 160UV/Vis (Thermo Scientific). A peak in the range of 250-300 nm isattributed to BHT stabilizer present in the raw material (poloxamer).Table 8 lists physicochemical properties of the above solutions beforeand after filtration.

TABLE 8 Physicochemical properties of 17% poloxamer 407 placebo solutionbefore and after filtration Viscosity^(a) @ 19° C. Absorbance @ SampleTgel (° C.) (cP) 274 nm Before filtration 22 100 0.3181 After filtration22 100 0.3081 ^(a)Viscosity measured at a shear rate of 37.5 s⁻¹

The above process is applicable for manufacture of 17% P407formulations, and includes temperature analysis of the room conditions.Preferably, a maximum temperature of 19° C. reduces cost of cooling thecontainer during manufacturing. In some instances, a jacketed containeris used to further control the temperature of the solution to easemanufacturing concerns.

Example 21 In Vitro Release of Otic Agent from an Autoclaved MicronizedSample

17% poloxamer 407/1.5% otic agent in TRIS buffer: 250.8 mg of sodiumchloride (Fisher Scientific), and 302.4 mg of Tromethamine (SigmaChemical Co.) is dissolved in 39.3 g of sterile filtered DI water, pH isadjusted to 7.4 with 1M HCl. 4.9 g of the above solution is used and anappropriate amount of micronized otic agent is suspended and dispersedwell. 2 mL of the formulation is transferred into a 2 mL glass vial(Wheaton serum glass vial) and sealed with 13 mm butyl styrene (kimblestoppers) and crimped with a 13 mm aluminum seal. The vial is placed ina Market Forge-sterilmatic autoclave (settings, slow liquids) andsterilized at 250° F. for 25 minutes. After the autoclaving the sampleis left to cool down to room temperature. The vial is placed in therefrigerator and mixed while cold to homogenize the sample. Samplediscoloration or precipitation after autoclaving is recorded.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm), 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL PBS buffer isplaced into reservoir and shaken using a Labline orbit shaker at 70 rpm.Samples are taken every hour [0.1 mL withdrawn and replaced with warmPBS buffer containing 2% PEG-40 hydrogenated castor oil (BASF) toenhance otic agent solubility]. Samples are analyzed for otic agentconcentration by UV at 245 nm against an external calibration standardcurve. The release rate is compared to other formulations disclosedherein. MDT time is calculated for each sample.

Solubilization of otic agent in the 17% poloxamer system is evaluated bymeasuring the concentration of the otic agent in the supernatant aftercentrifuging samples at 15,000 rpm for 10 minutes using an eppendorfcentrifuge 5424. Otic agent concentration in the supernatant is measuredby UV at 245 nm against an external calibration standard curve.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, prepared according to the procedures describedherein, are tested using the above procedures to determine release rateof the otic agent from each formulation.

Example 22 Release Rate or MDT and Viscosity of Formulation ContainingSodium Carboxymethyl Cellulose

17% poloxamer 407/2% otic agent/1% CMC (Hercules Blanose 7M): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.0) in PBS buffer is preparedby dissolving 205.6 mg of sodium chloride (Fisher Scientific), 372.1 mgof sodium phosphate dibasic dihydrate (Fisher Scientific), 106.2 mg ofsodium phosphate monobasic monohydrate (Fisher Scientific) in 78.1 g ofsterile filtered DI water. 1 g of Blanose 7M CMC (Hercules, viscosity of533 cP @ 2%) is sprinkled into the buffer solution and heated to easesolution, solution is then cooled down and 17.08 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. Aformulation comprising 17% poloxamer 407NF/1% CMC/2% otic agent in PBSbuffer is made adding/dissolving an appropriate amount of otic agent to9.8 g of the above solution, and mixing until all the otic agent iscompletely dissolved.

17% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7M65): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.2) in PBS buffer is preparedby dissolving 257 mg of sodium chloride (Fisher Scientific), 375 mg ofsodium phosphate dibasic dihydrate (Fisher Scientific), 108 mg of sodiumphosphate monobasic monohydrate (Fisher Scientific) in 78.7 g of sterilefiltered DI water. 0.502 g of Blanose 7M65 CMC (Hercules, viscosity of5450 cP @ 2%) is sprinkled into the buffer solution and heated to easesolution, solution is then cooled down and 17.06 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. A17% poloxamer 407NF/1% CMC/2% otic agent solution in PBS buffer is madeadding/dissolving an appropriate amount of otic agent to 9.8 g of theabove solution, and mixing until the otic agent is completely dissolved.

17% poloxamer 407/2% otic agent/0.5% CMC (Blanose 7H9): A sodiumcarboxymethylcellulose (CMC) solution (pH 7.3) in PBS buffer is preparedby dissolving 256.5 mg of sodium chloride (Fisher Scientific), 374 mg ofsodium phosphate dibasic dihydrate (Fisher Scientific), 107 mg of sodiumphosphate monobasic monohydrate (Fisher Scientific) in 78.6 g of sterilefiltered DI water, then 0.502 g of Blanose 7H9 CMC (Hercules, viscosityof 5600 cP @ 1%) is sprinkled to the buffer solution and heated to easesolution, solution is then cooled down and 17.03 g poloxamer 407NF(Spectrum Chemicals) is sprinkled into the cold solution while mixing. A17% poloxamer 407NF/1% CMC/2% otic agent solution in PBS buffer is madeadding/dissolving an appropriate amount of otic agent to 9.8 of theabove solution, and mixing until the otic agent is completely dissolved.

Viscosity measurements are performed using a Brookfield viscometerRVDV-II+P with a CPE-40 spindle rotated at 0.08 rpm (shear rate of 0.6s⁻¹), equipped with a water jacketed temperature control unit(temperature ramped from 10-34° C. at 1.6° C./min). Tgel is defined asthe inflection point of the curve where the increase in viscosity occursdue to the sol-gel transition.

Dissolution is performed at 37° C. in snapwells (6.5 mm diameterpolycarbonate membrane with a pore size of 0.4 μm). 0.2 mL of gel isplaced into snapwell and left to harden, then 0.5 mL PBS buffer isplaced into reservoir and shaken using a Labline orbit shaker at 70 rpm.Samples are taken every hour, 0.1 mL withdrawn and replaced with warmPBS buffer. Samples are analyzed for otic agent concentration by UV at245 nm against an external calibration standard curve. The release rateis compared to the formulations disclosed in above examples, and MDTtime is calculated for each of the above formulations.

Formulations comprising DNQX, D-methionine, micronized AM-101 ormicronized (S)-ketamine, prepared according to procedures describedabove, are tested using the above procedures to determine relationshipbetween release rate and/or mean dissolution time and viscosity offormulation containing sodium carboxymethyl cellulose. Any correlationbetween the mean dissolution time (MDT) and the apparent viscosity(measured at 2° C. below the gelation temperature) is recorded.

Example 23 Effect of Poloxamer Concentration and Otic AgentConcentration on Release Kinetics

A series of compositions comprising varying concentrations of a gellingagent and micronized dexamethasone was prepared using proceduresdescribed above. The mean dissolution time (MDT) for each composition inTable 9 was determined using procedures described above.

TABLE 9 Preparation of poloxamer/otic agent compositions Sample pH MDT15.5% P407/1.5% dexamethasone/PBS 7.4 46 h   16% P407/1.5%dexamethasone/PBS 7.4 40 h   17% P407/1.5% dexamethasone/PBS 7.4 39 h15.5% P407/4.5% dexamethasone/PBS 7.4 >7 days   16% P407/4.5%dexamethasone/PBS 7.4 >7 days   17% P407/4.5% dexamethasone/PBS 7.4 >7days

The effect of gel strength and otic agent concentration on releasekinetics of an otic agent from the composition or device was determinedby measurement of the MDT for poloxamer, and measurement of MDT for oticagent. The half life of the otic agent and mean residence time of theotic agent was also determined for each formulation by measurement ofconcentration of the otic agent in the perilymph.

The apparent viscosity of each composition was measured as describedabove. A thermoreversible polymer gel concentration of about 15.5% in acomposition or device described above provided an apparent viscosity ofabout 270,000 cP. A thermoreversible polymer gel concentration of about16% in a composition or device described above provided an apparentviscosity of about 360,000 cP. A thermoreversible polymer gelconcentration of about 17% in a composition or device described aboveprovided an apparent viscosity of about 480,000 cP.

Compositions comprising micronized DNQX, D-methionine, micronized AM-101or micronized (S)-ketamine, prepared according to the proceduresdescribed above, are tested using the above procedure to determinerelease rate of the otic agent from each composition.

Example 24 Application of an Enhanced Viscosity Auris Sensory CellModulating Agent Formulation onto the Round Window Membrane

A formulation comprising AM-101 prepared according to Example 8 isprepared and loaded into 5 ml siliconized glass syringes attached to a15-gauge luer lock disposable needle. Lidocaine is topically applied tothe tympanic membrane, and a small incision made to allow visualizationinto the middle ear cavity. The needle tip is guided into place over theround window membrane, and the auris sensory cell modulating agentformulation applied directly onto the round-window membrane.

Example 25 In Vivo Testing of Intratympanic Injection of Auris SensoryCell Modulating Agent Formulation in a Guinea Pig

A cohort of 21 guinea pigs (Charles River, females weighing 200-300 g)is intratympanically injected with 50 μL of different P407-otic agentformulations described herein, containing 0 to 50% otic agent. The gelelimination time course for each formulation is determined. A faster gelelimination time course of a formulation indicates lower meandissolution time (MDT). Thus the injection volume and the concentrationof an auris sensory cell modulating agent in a formulation are tested todetermine optimal parameters for preclinical and clinical studies.

Example 26 In Vivo Extended Release Kinetics

A cohort of 21 guinea pigs (Charles River, females weighing 200-300 g)is intratympanically injected with 50 μL 17% Pluronic F-127 formulationbuffered at 280 mOsm/kg and containing 1.5% to 35% auris sensory cellmodulating agent by weight of the formulation. Animals are dosed onday 1. The release profile for the formulations is determined based onanalysis of the perilymph.

Example 27 Evaluation of (S)-Ketamine in a Tinnitus Mouse Model

Twelve Harlan Sprague-Dawley mice weighing 20 to 24 g are used. Eachmouse is trained to drink from a water dispenser during periods nosound, but to refrain from drinking in the presence of sound. Each mouseis administered 350 mg of aspirin per kilogram of body weight (mg/kg).

The control group (n=10) are administered saline followingadministration of the aspirin. The experimental group (n=10) areadministered (S)-ketamine (400 mg/kg of body weight) followingadministration of the aspirin. Administration occurs via anintra-tympanic injection.

Following administration of (S)-ketamine whether the mice drink in theabsence of external sound is monitored.

Example 28 Evaluation of N-Acetylcysteine (NAC) in a Cisplatin-InducedOtotoxicity Mouse Model

Methods and Materials

Induction of Ototoxicity

Twelve Harlan Sprague-Dawley mice weighing 20 to 24 g are used. Baselineauditory brainstem response (ABR) at 4-20 mHz is measured. The mice aretreated with cisplatin (6 mg/kg of body weight). The cisplatin isdelivered to the aorta by IV infusion.

Treatment

The control group (n=10) are administered saline followingadministration of the cisplatin. The experimental group (n=10) areadministered NAC (400 mg/kg of body weight) following administration ofthe cisplatin.

Analysis of Results

Electrophysiologic Testing

The hearing threshold for the auditory brainstem response threshold(ABR) to click stimuli for each ear of each animal is initially measuredand 1 week after the experimental procedure. The animals are placed in asingle-walled acoustic booth (Industrial Acoustics Co, Bronx, N.Y., USA)on a heating pad. Subdermal electrodes (Astro-Med, Inc. Grass InstrumentDivision, West Warwick, R.I., USA) were inserted at the vertex (activeelectrode), the mastoid (reference), and the hind leg (ground). Clickstimuli (0.1 millisecond) are computer generated and delivered to aBeyer DT 48, 200 Ohm speaker fitted with an ear speculum for placementin the external auditory meatus. The recorded ABR is amplified anddigitized by a battery-operated preamplifier and input to a Tucker-DavisTechnologies ABR recording system that provides computer control of thestimulus, recording, and averaging functions (Tucker Davis Technology,Gainesville, Fla., USA). Successively decreasing amplitude stimuli arepresented in 5-dB steps to the animal, and the recorded stimulus-lockedactivity is averaged (n=512) and displayed. Threshold is defined as thestimulus level between the record with no visibly detectable responseand a clearly identifiable response.

Example 29 Clinical Trial of (S)-Ketamine as a Treatment for Tinnitus

Active Ingredient: (S)-ketamine

Dosage: 10 ng delivered in 10 μL of a thermoreversible gel. Release of(S)-ketamine is controlled release and occurs over thirty (30) days.

Route of Administration: Intratympanic injection

Treatment Duration: 12 weeks

Methodology

Monocentric

Prospective

Randomized

Double-blind

Placebo-controlled

Parallel group

Adaptive

Inclusion Criteria

Male and female subjects between the 18 and 64 years of age.

Subjects experiencing subjective tinnitus.

Duration of tinnitus is greater than 3 months.

No treatment of tinnitus within 4 weeks.

Evaluation Criteria

Efficacy (Primary)

-   -   1. Total score of the Tinnitus Questionnaire

Efficacy (Secondary)

-   -   1. Audiometric measurements (mode, frequency, loudness of the        tinnitus, pure tone audiogram, speech audiogram)    -   2. Quality of Life questionnaire

Safety

-   -   1. Treatment groups were compared with respect to incidence        rates of premature termination, treatment-emergent adverse        events, laboratory abnormalities, and ECG abnormalities.

Study Design

Subjects are divided into three treatment groups. The first group is thesafety sample. The second group is the intent-to-treat (ITT) sample. Thethird group is the valid for efficacy (VfE) group.

For each group, one half of subjects to be given (S)-ketamine and theremainder to be given placebo.

Statistical Methods

The primary efficacy analysis is based on the total score of theTinnitus Questionnaire in the ITT sample. The statistical analysis isbased on an analysis of covariance (ANCOVA) with baseline as covariantand the last observation carried forward value as dependent variable.Factor is “treatment.” The homogeneity of regression slopes is tested.The analysis is repeated for the VfE sample.

Audiometric measurements (mode, frequency, loudness of the tinnitus,pure tone audiogram, speech audiogram) as well as quality of life arealso analyzed via the aforementioned model. The appropriateness of themodel is not tested. P values are exploratory and are not adjusted formultiplicity.

Example 30 Evaluation of AMN082 on Cisplatin-Induced Ototoxicity

Study Objective

The primary objective of this study will be to assess the safety andefficacy of AMN082 (100 mg) compared with that of placebo in preventingCisplatin-induced ototoxicity.

Methods

Study Design

This will be a phase 3, multicentre, double-blind, randomised,placebo-controlled, parallel group study comparing AMN082 (100 mg) toplacebo in the treatment of Cisplatin-induced ototoxicity. Approximately140 subjects will be enrolled in this study, and randomised (1:1) to 1of 2 treatment groups based on a randomisation sequence prepared bysponsor. Each group will receive either AMN082 100 mg or placebo.

Subjects who do not complete the study will not be replaced. Patientswill receive weekly chemotherapy (cisplatin at a dose of 70 mg/m² for 7weeks and daily radiation. Following chemotherapy, patients will receivethe study drug (AMN082 500 mg or matching placebo) administered as a gelformulation directly onto the subjects' round window membrane for 8weeks.

Each patient will receive a hearing evaluation before each treatmentwith Cisplatin. Two to four weeks after the final dose of Cisplatin,each patient will receive a hearing evaluation. Pre-treatment audiogramwill be compared with the post treatment audiogram to determine thedegree of cisplatin-induced ototoxicity. Patients will thereafterreceive a hearing evaluation at 4-week intervals concomitant with theAMN082 treatment.

Main Criteria for Inclusion

Male or female outpatients aged between 18 and 75 years receivingchemotherapy with Cisplatin. Patients expected to receive a minimum of 3rounds of chemotherapy. If a subject becomes pregnant during the study,she will be immediately withdrawn and no study medication will beadministered.

Exclusion Criteria

Patients who have had middle ear surgery. Patients who have activeexternal or middle ear disease. Patients who have preceding pure toneaverage of >40 dB HL.

Example 31 Clinical Trial of AM-101 as a Treatment for Noise InducedHearing Loss

Active Ingredient: AM-101

Dosage: A composition comprising 4% by weight of micronized AM-101delivered in 10 μL dose of a thermoreversible gel. Release of AM-101 iscontrolled release and occurs over 3 weeks.

Route of Administration: Intratympanic injection

Treatment Duration: 12 weeks, one injection every 3 weeks

Methodology

Monocentric

Prospective

Randomized

Double-blind

Placebo-controlled

Parallel group

Adaptive

Inclusion Criteria

-   -   Male and female subjects between the 18 and 64 years of age.    -   Acoustic trauma followed by hearing loss that is documented by        audiogram and medical report with an inner ear hearing loss of        at least 15 dB    -   Acute tinnitus that has persisted for at least 3 months.    -   No prior treatment of tinnitus within 4 weeks.

Evaluation Criteria

Efficacy (Primary)

-   -   1. Total score of the Tinnitus Questionnaire

Efficacy (Secondary)

-   -   1. Audiometric measurements (mode, frequency, loudness of the        tinnitus, pure tone audiogram, speech audiogram)    -   2. Quality of Life questionnaire

Safety

-   -   1. Treatment groups were compared with respect to incidence        rates of premature termination, treatment-emergent adverse        events, laboratory abnormalities, and ECG abnormalities.

Study Design

Subjects are divided into three treatment groups. The first group is thesafety sample. The second group is the intent-to-treat (ITT) sample. Thethird group is the valid for efficacy (VfE) group.

For each group, one half of subjects to be given AM-101 and theremainder to be given placebo.

Statistical Methods

The primary efficacy analysis is based on the total score of theTinnitus Questionnaire in the ITT sample. The statistical analysis isbased on an analysis of covariance (ANCOVA) with baseline as covariantand the last observation carried forward value as dependent variable.Factor is “treatment.” The homogeneity of regression slopes is tested.The analysis is repeated for the VfE sample.

Audiometric measurements (mode, frequency, loudness of the tinnitus,pure tone audiogram, speech audiogram) as well as quality of life arealso analyzed via the aforementioned model. The appropriateness of themodel is not tested. P values are exploratory and are not adjusted formultiplicity.

Example 32 Clinical Trial of (S)-Ketamine as a Treatment in Combinationwith Implantation of a Cochlear Hearing Device

Active Ingredient: (S)-ketamine in combination with dexamethasone

Dosage: A composition comprising micronized (S)-ketamine and micronizeddexamethasone, used as a pre-surgical irrigation solution and apost-surgical irrigation solution. Release of (S)-ketamine anddexamethasone is immediate release.

Study Design

Twenty patients will be enrolled in the study. Ten patients will be inthe control group and ten patients will be in the treatment group.

Eligibility criteria

-   -   having severe to profound sensorineural hearing impairment in        both ears    -   having a functioning auditory nerve    -   having lived at least a short amount of time without hearing        (approximately 70+ decibel hearing loss, on average)    -   having good speech, language, and communication skills, or in        the case of infants and young children, having a family willing        to work toward speech and language skills with therapy    -   not benefiting enough from other kinds of hearing aids    -   having no medical reason to avoid surgery

Each patient will be subjected to chochloestomy and insertion ofelectrodes. The treatment group will be subjected to perfusion of thesurgical area with the test composition prior to surgery and aftersurgery. The patients will be monitored for 6 weeks. Intracochleartrauma will be evaluated based on audiometric measurements, speechaudiogram as well as quality of life. Occurrence of secondary infectionsand/or inflammation will be monitored.

Example 33 Clinical Trial of Auris Sensory Cell Modulator Formulationsin Combination with Surgery

The purpose of this study is to determine if a composition comprising acombination of AM-101 and Dexamethasone administered in combination withtympanostomy is safe and effective in preventing and/or treating middleear infections in patients with ear tubes.

Study Type: Interventional

Study Design: This will be a non-inferiority open label study to comparethe current standard of care versus the use of extended releaseintratympanic compositions in combination with tympanostomy. The currentstandard of care requires the use of otic drops for 5-7 dayspost-surgery. The study is designed to test whether administration of asustained release composition at the time of surgery obviates the needfor out-patient treatment. The test hypothesis is that administration ofa single injection of an extended release composition at the time ofsurgery is not inferior to administration of otic drops after surgery.

Inclusion Criteria:

-   -   6 months to 12 years old, hearing loss in one or both ears    -   Patient may not have had otic surgery other than tube placement        in the last year    -   Patient may not have any disease or condition that would        negatively affect the conduct of the study    -   Patient may not require any other systemic antimicrobial therapy        during the study.    -   Analgesic use (other than acetaminophen) is not allowed

Exclusion Criteria: Age

Study Protocol: Twenty patients will be divided into two groups. Thefirst group of patients will receive an injection of an extended releasecomposition comprising micronized AM-101 and micronized dexamethasone,prepared according to Example 22, during the surgical procedure. Eachpatient will undergo a tympanostomy for placement of a tube. During thesurgical procedure, the surgeon will clean the ear and while themyngotomoy incision is open, the surgeon injects a test composition intothe middle ear space. The tube is inserted after injection of theextended release composition into the middle ear space. The testcomposition is either prepared in the operating room by suspending drymicronized powder of AM-101 and dexamethasone with other excipients, orthe test composition is a prepared suspension ready for injection.

The second group of patients will be given ear drops comprisingnon-micronized AM-101 and non-micronized dexamethasone as immediaterelease components to be administered for 5-7 days after the surgery.

Patients are monitored with weekly follow up visits for one month. Anydifferences in treatment outcomes between the two groups are recorded.

Primary Outcome Measures: Time to cessation of otorrhea as recorded bythe parent or guardian via a patient.

Secondary Outcome Measures: Clinical cure rate; Microbiological outcome;Treatment failures; Recurrence of disease.

The treatment outcome for each group of patients is compared todetermine whether administration of the extended release compositioncomprising AM-101 and dexamethasone in combination with tympanostomy isno worse than administration of ear drops comprising AM-101 anddexamethasone after surgery for reduction of otorrhea, infections,and/or inflammation associated with tympanostomy.

While preferred embodiments of the present invention have been shown anddescribed herein, such embodiments are provided by way of example only.Various alternatives to the embodiments described herein are optionallyemployed in practicing the inventions. It is intended that the followingclaims define the scope of the invention and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

1-22. (canceled)
 23. A pharmaceutical formulation for use in thetreatment of an otic disease or condition formulated to provide to theinner ear of a patient in need thereof a therapeutically-effective,sustained-release amount of an agent that modulates the Atoh1 gene, theformulation comprising: between about 0.2% to about 20% by weight of theagent that modulates the Atoh1 gene or pharmaceutically acceptableprodrug or salt thereof; and a thermoreversible gel having a gelationtemperature between about 19° C. and about 40° C.
 24. The formulation ofclaim 23, wherein the agent comprises a vector engineered to carry thehuman Atoh1 gene.
 25. The formulation of claim 23, wherein the agentthat modulates the Atoh 1 gene is an Atoh 1 polypeptide.
 26. Theformulation of claim 23, wherein the agent that modulates the Atohl geneinduces auris sensory hair cell regeneration.
 27. The formulation ofclaim 23, wherein the agent that modulates the Atohl gene is releasedfrom the formulation for a period of at least 3 days.
 28. Theformulation of claim 23, wherein the thermoreversible gel comprises apolymer composed of polyoxypropylene and polyoxyethylene.
 29. Theformulation of claim 23, wherein the thermoreversible gel comprisesbetween about 16% to about 21% by weight of polymers composed ofpolyoxypropylene and polyoxyethylene.
 30. The formulation of claim 23,wherein the otic disease or condition is hearing loss.
 31. Theformulation of claim 30, wherein the hearing loss is from Meniere'sdisease, sudden sensorineural hearing loss, noise induced hearing loss,age related hearing loss, auto immune ear disease or tinnitus.
 32. Amethod for treating an otic disease or condition comprisingadministering to a patient in need thereof a sustained releasepharmaceutical composition formulated to provide to the inner ear of apatient in need thereof a therapeutically-effective amount of an agentthat modulates the Atoh1 gene, the formulation comprising: between about0.2% to about 20% by weight of the agent that modulates the Atoh1 geneor pharmaceutically acceptable prodrug or salt thereof; and athermoreversible gel having a gelation temperature between about 19° C.and about 40° C.
 33. The method of claim 32, wherein the agent comprisesa vector engineered to carry the human Atoh1 gene.
 34. The method ofclaim 32, wherein the agent that modulates the Atoh1 gene is an Atoh1polypeptide.
 35. The method of claim 32, wherein the agent thatmodulates the Atohl gene induces auris sensory hair cell regeneration.36. The method of claim 32, wherein the agent that modulates the Atohlgene is released from the formulation for a period of at least 3 days.37. The method of claim 32, wherein the thermoreversible gel comprises apolymer composed of polyoxypropylene and polyoxyethylene.
 38. The methodof claim 32, wherein the thermoreversible gel comprises between about16% to about 21% by weight of polymers composed of polyoxypropylene andpolyoxyethylene.
 39. The method of claim 32, wherein the otic disease orcondition is hearing loss.
 40. The method of claim 39, wherein thehearing loss is from Meniere's disease, sudden sensorineural hearingloss, noise induced hearing loss, age related hearing loss, auto immuneear disease or tinnitus.
 41. The method of claim 32, wherein thepharmaceutical composition is administered at or near the round windowmembrane of the patient in need.